Oxidative Stress Contributes to Endothelial Dysfunction in Mouse Models of Hereditary Hemorrhagic Telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase (ALK1; HHT2) genes, coding for transforming growth factor-β(TGF-β) superfamily receptors. We demonstrated previously that endoglin and ALK1 interact with endothelial NO synthase (eNOS) and affect its activation. Endothelial cells deficient in endoglin or ALK1 proteins show eNOS uncoupling, reduced NO, and increased reactive oxygen species (ROS) production. In this study, we measured NO and H2O2levels in several organs of adultEngandAlk1heterozygous mice, to ascertain whether decreased NO and increased ROS production is a generalized manifestation of HHT. A significant reduction in NO and increase in ROS production were found in several organs, known to be affected in patients. ROS overproduction in mutant mice was attributed to eNOS, as it was L-NAME inhibitable. Mitochondrial ROS contribution, blocked by antimycin, was highest in liver while NADPH oxidase, inhibited by apocynin, was a major source of ROS in the other tissues. However, there was no difference in antimycin- and apocynin-inhibitable ROS production between mutant and control mice. Our results indicate that eNOS-derived ROS contributes to endothelial dysfunction and likely predisposes to disease manifestations in several organs of HHT patients.

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Reactive oxygen species cause endothelial dysfunction in chronic flow overload

Journal of Applied Physiology ◽

10.1152/japplphysiol.00786.2009 ◽

2011 ◽

Vol 110 (2) ◽

pp. 520-527 ◽

Cited By ~ 17

Author(s):

X. Lu ◽

X. Guo ◽

C. D. Wassall ◽

M. D. Kemple ◽

J. L. Unthank ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Shear Stress ◽

Endothelial Dysfunction ◽

Nadph Oxidase ◽

Group Treatment ◽

Carotid Arteries ◽

Reactive Oxygen ◽

Ros Production ◽

Oxygen Species ◽

Enos Uncoupling

Although elevation of shear stress increases production of vascular reactive oxygen species (ROS), the role of ROS in chronic flow overload (CFO) has not been well investigated. We hypothesize that CFO increases ROS production mediated in part by NADPH oxidase, which leads to endothelial dysfunction. In six swine, CFO in carotid arteries was induced by contralateral ligation for 1 wk. In an additional group, six swine received apocynin (NADPH oxidase blocker and anti-oxidant) treatment in conjunction with CFO for 1 wk. The blood flow in carotid arteries increased from 189.2 ± 25.3 ml/min (control) to 369.6 ± 61.9 ml/min (CFO), and the arterial diameter increased by 8.6%. The expressions of endothelial nitric oxide synthase (eNOS), p22/p47phox, and NOX2/NOX4 were upregulated. ROS production increased threefold in response to CFO. The endothelium-dependent vasorelaxation was compromised in the CFO group. Treatment with apocynin significantly reduced ROS production in the vessel wall, preserved endothelial function, and inhibited expressions of p22/p47phox and NOX2/NOX4. Although the process of CFO remodeling to restore the wall shear stress has been thought of as a physiological response, the present data implicate NADPH oxidase-produced ROS and eNOS uncoupling in endothelial dysfunction at 1 wk of CFO.

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Abstract 538: Overexpression of the Catalytic Subunit of Telomerase Protects Against Ang II Induced Vascular Dysfunction

Hypertension ◽

10.1161/hyp.64.suppl_1.538 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Joe Hockenberry ◽

David D Gutterman ◽

Andreas M Beyer

Keyword(s):

Endothelial Dysfunction ◽

Vascular Dysfunction ◽

Catalytic Subunit ◽

Flow Mediated Dilation ◽

Ros Production ◽

Mitochondrial Ros ◽

Ros Formation ◽

Tissue Aging ◽

And Control ◽

Mini Pump

Rational: Angiotensin II (A II) produces endothelial dysfunction by elevating vascular reactive oxygen species (ROS) production from mitochondria. Telomerase, prominent in cellular senescence and tissue aging, reduces mitochondrial ROS production. Conversely, loss of the catalytic subunit of telomerase (TERT-/-) causes hypertension and reduced NO bioavailability. We have shown previously that decreased TERT abrogates NO mediated dilation and increases mtROS levels. We hypothesized that overexpression of TERT attenuates A II induced endothelial dysfunction by suppressing ROS production, while loss of TERT predisposes to A II induced endothelial dysfunction. Results: Mouse mesenteric arterioles (~250 μm) from hTERT transgenic, TERT-/- and control mice were infused with A II (via osmotic mini pump, 2 weeks) were used. Flow-mediated dilation (FMD) to graded degrees of shear was measured in vessels constricted with norepinephrine. A II (400ng/kg/min) reduced FMD in TERT-/- but not in WT controls (left). The mechanism of FMD was changed in TERT-/- from NO to H2O2 as previously shown. TERT transgenic mice showed hyper relaxation to shear which was normalized by A II treatment (1000ng/kg/min), while in WT control animals the same dose of A II reduced endothelium-dependent dilation (right). Conclusions: We conclude that up-regulation of TERT is sufficient to prevent A II mediated endothelial dysfunction, likely by suppressing ROS formation and preserving physiological NO levels in the microvascular.

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Nitrogen Dioxide Inhalation Exposures Induce Cardiac Mitochondrial Reactive Oxygen Species Production, Impair Mitochondrial Function and Promote Coronary Endothelial Dysfunction

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17155526 ◽

2020 ◽

Vol 17 (15) ◽

pp. 5526

Author(s):

Ahmed Karoui ◽

Clément Crochemore ◽

Najah Harouki ◽

Cécile Corbière ◽

David Preterre ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Risk Factor ◽

Endothelial Dysfunction ◽

Mitochondrial Function ◽

Reactive Oxygen ◽

Ros Production ◽

Oxygen Species ◽

Mitochondrial Reactive Oxygen Species ◽

Mitochondrial Ros ◽

Coronary Endothelial Dysfunction

Traffic air pollution is a major health problem and is recognized as an important risk factor for cardiovascular (CV) diseases. In a previous experimental study, we showed that diesel exhaust (DE) exposures induced cardiac mitochondrial and CV dysfunctions associated with the gaseous phase. Here, we hypothesized that NO2 exposures to levels close to those found in DE induce a mitochondrial reactive oxygen species (ROS) production, which contribute to an endothelial dysfunction, an early indicator for numerous CV diseases. For this, we studied the effects of NO2 on ROS production and its impacts on the mitochondrial, coronary endothelial and cardiac functions, after acute (one single exposure) and repeated (three h/day, five days/week for three weeks) exposures in Wistar rats. Acute NO2 exposure induced an early but reversible mitochondrial ROS production. This event was isolated since neither mitochondrial function nor endothelial function were impaired, whereas cardiac function assessment showed a reversible left ventricular dysfunction. Conversely, after three weeks of exposure this alteration was accompanied by a cardiac mitochondrial dysfunction highlighted by an alteration of adenosine triphosphate (ATP) synthesis and oxidative phosphorylation and an increase in mitochondrial ROS production. Moreover, repeated NO2 exposures promoted endothelial dysfunction of the coronary arteries, as shown by reduced acetylcholine-induced vasodilatation, which was due, at least partially, to a superoxide-dependent decrease of nitric oxide (NO) bioavailability. This study shows that NO2 exposures impair cardiac mitochondrial function, which, in conjunction with coronary endothelial dysfunction, contributes to cardiac dysfunction. Together, these results clearly identify NO2 as a probable risk factor in ischemic heart diseases.

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A Novel Missense Mutation in the Endoglin Gene in Hereditary Hemorrhagic Telangiectasia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655946 ◽

1997 ◽

Vol 77 (02) ◽

pp. 243-247 ◽

Cited By ~ 15

Author(s):

Hiroshi Yamaguchi ◽

Hiroyuki Azuma ◽

Toshio Shigekiyo ◽

Hideo Inoue ◽

Shiro Saito

Keyword(s):

Missense Mutation ◽

Hereditary Hemorrhagic Telangiectasia ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Autosomal Dominant Disorder ◽

Her Family ◽

Normal Individuals ◽

Oligonucleotide Hybridization ◽

Vascular Dysplasia ◽

Exon 4

SummaryHereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystem vascular dysplasia and recurrent hemorrhage. Recent investigation has mapped one of the responsible genes for HHT to chromosome 9q33-q34; subsequently, nine different mutations have been identified in the endoglin gene, which encodes a transforming growth factor β(TGF-β) binding protein, in nine unrelated families with HHT. We examined the endoglin gene in a Japanese patient with HHT and her family members. Using PCR-SSCP. analysis followed by sequencing, we identified a C to A missense mutation in exon 4 which changed an Ala160 codon(GCT) to an Asp160 codon (GAT). Since this mutation destroys one of three Fnu4H I sites in exon 4, the Fnu4H I digestion patterns of the PCR-amplified exon 4 fragments from each family member were analyzed. In affected members, the restriction patterns were all consistent with a phenotype of HHT. PCR-amplified exon 4 fragments from 150 normal individuals were also analyzed by allele-specific oligonucleotide hybridization analysis. As a result, the mutation was not found in any of them. We conclude that the C to A mutation in exon 4 of the endoglin gene in this proband is responsible for the occurrence of HHT in this family.

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Abstract 252: 6ß-Hydroxytestosterone, A Cytochrome P450 1B1 Metabolite of Testosterone, Enhances Angiotensin II-Induced Pressor Effect in Male Mice

Hypertension ◽

10.1161/hyp.62.suppl_1.a252 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Ajeeth K Pingili ◽

Brett L Jennings ◽

Nayaab S Khan ◽

Kafait U Malik

Keyword(s):

Cytochrome P450 ◽

Endothelial Dysfunction ◽

Ros Production ◽

Oxygen Species ◽

Pressor Effect ◽

Ang Ii ◽

Male Mice ◽

Cytochrome P450 1B1 ◽

Induced Hypertension ◽

Novel Target

Androgens have been implicated in the development of hypertension and castration minimizes the pressor effect of angiotensin (Ang) II. Previously we showed that Ang II-induced hypertension and associated pathophysiological changes are diminished in male cytochrome P450 (CYP) 1B1 gene disrupted mice. Since CYP1B1 metabolizes testosterone to 6β-hydroxytestosterone (6β-OHT); this study was conducted to determine its contribution in modulation of Ang II-induced hypertension. Eight weeks old male Cyp1b1+/+ and Cyp1b1-/- mice were either castrated or injected with 6β-OHT (15 μg/g, i.p. every 3rd day) or vehicle (DMSO, 50 μl), infused with Ang II (700 ng/kg/min) or vehicle for 2 weeks, and systolic blood pressure (SBP) was measured by tail cuff. Castration attenuated Ang II-induced increase in SBP in both Cyp1b1+/+ (184 ± 6 vs. 129 ± 4 mmHg, P < 0.05) and Cyp1b1-/- mice (150 ± 6 vs. 129 ± 4 mmHg, P < 0.05). In Cyp1b1+/+ mice, 6β-OHT did not alter Ang II-induced increase in SBP (184 ± 6 vs. 180 ± 8 mmHg, P < 0.05), but enhanced it in Cyp1b1-/- mice (150 ± 6 vs. 172 ± 8 mmHg, P < 0.05). Castration improved endothelial dysfunction associated with Ang II-induced hypertension in Cyp1b1+/+ mice, as demonstrated by increased relaxation of the aorta to acetylcholine. No endothelial dysfunction was observed in Cyp1b1-/- mice given Ang II with or without castration. In Cyp1b1+/+ mice, 6β-OHT did not alter Ang II-induced endothelial dysfunction, however, in Cyp1b1-/- mice infused with Ang II, 6β-OHT caused endothelial dysfunction. We have shown that Ang II-induced hypertension is associated with increased vascular production of reactive oxygen species (ROS) in Cyp1b1+/+ mice, and this increase is attenuated in Cyp1b1-/- mice, as measured by dihydroethidium fluorescence. In both Cyp1b1+/+ and Cyp1b1-/- mice given Ang II, castration abolished the increased ROS production. In Cyp1b1+/+ mice, 6β-OHT did not alter levels of ROS produced by Ang II, however, 6β-OHT further increased ROS production in Cyp1b1-/- mice given Ang II. These data suggest that 6β-OHT, a CYP1B1 metabolite of testosterone, contributes to the hypertensive effect of Ang II in male mice. Moreover, CYP1B1 could serve as a novel target for the development of agents for the treatment of androgen-mediated hypertension.

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Ultraendurance exercise increases the production of reactive oxygen species in isolated mitochondria from human skeletal muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.00966.2009 ◽

2010 ◽

Vol 108 (4) ◽

pp. 780-787 ◽

Cited By ~ 47

Author(s):

Kent Sahlin ◽

Irina G. Shabalina ◽

C. Mikael Mattsson ◽

Linda Bakkman ◽

Maria Fernström ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Oxidative Damage ◽

Vastus Lateralis ◽

Reactive Oxygen ◽

Ros Production ◽

Oxygen Species ◽

Work Intensity ◽

Isolated Mitochondria ◽

Mitochondrial Ros

Exercise-induced oxidative stress is important for the muscular adaptation to training but may also cause muscle damage. We hypothesized that prolonged exercise would increase mitochondrial production of reactive oxygen species (ROS) measured in vitro and that this correlates with oxidative damage. Eight male athletes (24–32 yr) performed ultraendurance exercise (kayaking/running/cycling) with an average work intensity of 55% V̇o2peak for 24 h. Muscle biopsies were taken from vastus lateralis before exercise, immediately after exercise, and after 28 h of recovery. The production of H2O2 was measured fluorometrically in isolated mitochondria with the Amplex red and peroxidase system. Succinate-supported mitochondrial H2O2 production was significantly increased after exercise (73% higher, P = 0.025) but restored to the initial level at recovery. Plasma level of free fatty acids (FFA) increased fourfold and exceeded 1.2 mmol/l during the last 6 h of exercise. Plasma FFA at the end of exercise was significantly correlated to mitochondrial ROS production ( r = 0.74, P < 0.05). Mitochondrial content of 4-hydroxy-nonenal-adducts (a marker of oxidative damage) was increased only after recovery and was not correlated with mitochondrial ROS production. Total thiol group level and glutathione peroxidase activity were elevated after recovery. In conclusion, ultraendurance exercise increases ROS production in isolated mitochondria, but this is reversed after 28 h recovery. Mitochondrial ROS production was not correlated with oxidative damage of mitochondrial proteins, which was increased at recovery but not immediately after exercise.

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Mitochondrial Reactive Oxygen Species Mediate GPCR–induced TACE/ADAM17-dependent Transforming Growth Factor-α Shedding

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1256 ◽

2009 ◽

Vol 20 (24) ◽

pp. 5236-5249 ◽

Cited By ~ 48

Author(s):

Timothy J. Myers ◽

Leann H. Brennaman ◽

Mary Stevenson ◽

Shigeki Higashiyama ◽

William E. Russell ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Growth Factor ◽

Transforming Growth Factor ◽

Reactive Oxygen ◽

Oxygen Species ◽

Ros Scavenging ◽

Transforming Growth Factor Α ◽

Egfr Activation ◽

Mitochondrial Ros ◽

Factor Α

Epidermal growth factor receptor (EGFR) activation by GPCRs regulates many important biological processes. ADAM metalloprotease activity has been implicated as a key step in transactivation, yet the regulatory mechanisms are not fully understood. Here, we investigate the regulation of transforming growth factor-α (TGF-α) shedding by reactive oxygen species (ROS) through the ATP-dependent activation of the P2Y family of GPCRs. We report that ATP stimulates TGF-α proteolysis with concomitant EGFR activation and that this process requires TACE/ADAM17 activity in both murine fibroblasts and CHO cells. ATP-induced TGF-α shedding required calcium and was independent of Src family kinases and PKC and MAPK signaling. Moreover, ATP-induced TGF-α shedding was completely inhibited by scavengers of ROS, whereas calcium-stimulated shedding was partially inhibited by ROS scavenging. Hydrogen peroxide restored TGF-α shedding after calcium chelation. Importantly, we also found that ATP-induced shedding was independent of the cytoplasmic NADPH oxidase complex. Instead, mitochondrial ROS production increased in response to ATP and mitochondrial oxidative complex activity was required to activate TACE-dependent shedding. These results reveal an essential role for mitochondrial ROS in regulating GPCR-induced growth factor shedding.

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Mitochondrial reactive oxygen species contribute to high NaCl-induced activation of the transcription factor TonEBP/OREBP

AJP Renal Physiology ◽

10.1152/ajprenal.00378.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. F1169-F1176 ◽

Cited By ~ 40

Author(s):

Xiaoming Zhou ◽

Joan D. Ferraris ◽

Maurice B. Burg

Keyword(s):

Reactive Oxygen Species ◽

Transcription Factor ◽

Nuclear Translocation ◽

Reactive Oxygen ◽

Hek293 Cells ◽

Organic Osmolytes ◽

Ros Production ◽

Oxygen Species ◽

Hypertonic Stress ◽

Mitochondrial Ros

Hypertonicity activates the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), resulting in increased expression of genes involved in osmoprotective accumulation of organic osmolytes, including glycine betaine, and in increased expression of osmoprotective heat shock proteins. Our previous studies showed that high NaCl increases reactive oxygen species (ROS), which contribute to activation of TonEBP/OREBP. Mitochondria are a major source of ROS. The purpose of the present study was to examine whether mitochondria produce the ROS that contribute to activation of TonEBP/OREBP. We inhibited mitochondrial ROS production in HEK293 cells with rotenone and myxothiazol, which inhibit mitochondrial complexes I and III, respectively. Rotenone (250 nM) and myxothiazol (12 nM) reduce high NaCl-induced ROS over 40%, whereas apocynin (100 μM), an inhibitor of NADPH oxidase, and allopurinol (100 μM), an inhibitor of xanthine oxidase, have no significant effect. Rotenone and myxothiazol reduce high NaCl-induced increases in TonEBP/OREBP transcriptional activity (ORE/TonE reporter assay) and BGT1 (betaine transporter) mRNA abundance ranging from 53 to 69%. They inhibit high NaCl-induced TonEBP/OREBP transactivating activity, but not its nuclear translocation. Release of ATP into the medium on hypertonic stress has been proposed to be a signal that triggers cellular osmotic responses. However, we do not detect release of ATP into the medium or inhibition of high NaCl-induced ORE/TonE reporter activity by an ATPase, apyrase (20 U/ml), indicating that high NaCl-induced activation of TonEBP/OREBP is not mediated by release of ATP. We conclude that high NaCl increases mitochondrial ROS production, which contributes to the activation of TonEBP/OREBP by increasing its transactivating activity.

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Mitochondrial-targeted antioxidants protect skeletal muscle against immobilization-induced muscle atrophy

Journal of Applied Physiology ◽

10.1152/japplphysiol.00591.2011 ◽

2011 ◽

Vol 111 (5) ◽

pp. 1459-1466 ◽

Cited By ~ 127

Author(s):

Kisuk Min ◽

Ashley J. Smuder ◽

Oh-sung Kwon ◽

Andreas N. Kavazis ◽

Hazel H. Szeto ◽

...

Keyword(s):

Oxidative Stress ◽

Skeletal Muscle ◽

Muscle Atrophy ◽

Muscle Fibers ◽

Skeletal Muscle Atrophy ◽

Ros Production ◽

Oxygen Species ◽

Mitochondrial Ros ◽

Protease Activation ◽

Limb Immobilization

Prolonged periods of muscular inactivity (e.g., limb immobilization) result in skeletal muscle atrophy. Although it is established that reactive oxygen species (ROS) play a role in inactivity-induced skeletal muscle atrophy, the cellular pathway(s) responsible for inactivity-induced ROS production remain(s) unclear. To investigate this important issue, we tested the hypothesis that elevated mitochondrial ROS production contributes to immobilization-induced increases in oxidative stress, protease activation, and myofiber atrophy in skeletal muscle. Cause-and-effect was determined by administration of a novel mitochondrial-targeted antioxidant (SS-31) to prevent immobilization-induced mitochondrial ROS production in skeletal muscle fibers. Compared with ambulatory controls, 14 days of muscle immobilization resulted in significant muscle atrophy, along with increased mitochondrial ROS production, muscle oxidative damage, and protease activation. Importantly, treatment with a mitochondrial-targeted antioxidant attenuated the inactivity-induced increase in mitochondrial ROS production and prevented oxidative stress, protease activation, and myofiber atrophy. These results support the hypothesis that redox disturbances contribute to immobilization-induced skeletal muscle atrophy and that mitochondria are an important source of ROS production in muscle fibers during prolonged periods of inactivity.

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Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00189.2014 ◽

2014 ◽

Vol 307 (7) ◽

pp. H996-H1004 ◽

Cited By ~ 14

Author(s):

Ricardo Quarrie ◽

Daniel S. Lee ◽

Levy Reyes ◽

Warren Erdahl ◽

Douglas R. Pfeiffer ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Myocardial Dysfunction ◽

Ischemia Reperfusion ◽

Reactive Oxygen ◽

Ros Production ◽

Oxygen Species ◽

Sprague Dawley ◽

Mitochondrial Uncoupling ◽

Mitochondrial Ros ◽

Ros Formation

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H+ leak decreases ROS formation; it has been postulated that increasing H+ leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H+ leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H+ leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H+ leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H+ leak. H+ leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H+ leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H+ leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

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