Bruno Fink

Publications of Bruno Fink

• TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction.

  Authors: Nithya Mariappan, Carrie M Elks, Bruno Fink, Joseph Francis

  Free radical biology & medicine. 12/2008;

  Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction.Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O(2)(-)) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O(2)(-) and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague-Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 mumol/kg bw, orally), and TNF+ Tempol (Temp; 300 mumol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O(2)(-) production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.

• Angiotensinergic stimulation of vascular endothelium in mice causes hypotension, bradycardia, and attenuated angiotensin response.

  Authors: Ramaswamy Ramchandran, Takanobu Takezako, Yasser Saad, Linda Stull, Bruno Fink, Hirotsugu Yamada, Sergey Dikalov, David G Harrison, Christine Moravec, Sadashiva S Karnik

  Proceedings of the National Academy of Sciences of the United States of America. 01/2007; 103(50):19087-92.

  It is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle cells (VSMCs) in the vasculature and whether impairmentIt is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle cells (VSMCs) in the vasculature and whether impairment of this regulation in vivo contributes to hypertension. Delineation of the actions of Ang II through the type 1 receptor (AT1R) on ECs in the blood vessels has been a challenging problem because of the predominance of the AT1R functions in VSMCs that lie underneath the endothelium. We have obviated this limitation by generating transgenic (TG) mice engineered to target expression of the constitutively active N111G mutant AT1R only in ECs. In these TG mice, the enhanced angiotensinergic signal in ECs without infusion of Ang II resulted in hypotension and bradycardia. The pressor response to acute infusion of Ang II was significantly reduced. Increased expression of endothelial nitric oxide synthase and production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, cause these phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1R-selective antagonist. Our results imply that the Ang II action by means of EC-AT1R is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease.

• ESR techniques for the detection of nitric oxide in vivo as an index of endothelial function.

  Authors: Bruno Fink, Sergey Dikalov, Nelli Fink

  Pharmacological reports : PR. 02/2006; 58 Suppl:8-15.

  Plasma nitrite/nitrate levels reflect oxidation of formed nitric oxide (NO ) but are not indicative of endothelial nitric oxide synthase (eNOS) function due to interference by dietary nitrates andPlasma nitrite/nitrate levels reflect oxidation of formed nitric oxide (NO ) but are not indicative of endothelial nitric oxide synthase (eNOS) function due to interference by dietary nitrates and reactive oxygen species (ROS). Nitrosyl hemoglobin (NOHb), a metabolic product of nitric oxide, may better correlate with bioavailable NO but it may depend on the activity of different nitric oxide synthase (NOS) isoforms and may be affected by dietary nitrite/nitrate. We examined the correlation between vascular endothelial NO release and circulating blood levels of NOHb. We measured NOHb in blood using electron spin resonance (ESR) spectrometry and also quantified vascular production of NO using colloid Fe(DETC)(2) and ESR in mouse and human venous blood before and after treatment with the beta-blocker carvedilol. Exclusively the inhibition with L-NAME and not the treatment with the selective neuronal nitric oxide synthase (nNOS) inhibitor, N-AANG or with the selective inducible nitric oxide synthase (iNOS) inhibitor, 1400W, halved NOHb formation, which reflects the complete inhibition of NO release by aortic endothelium. The relationship between NOHb and NO production by the endothelium (0.23 microM NOHb to 3.73 microM/hour of NO per mg of aorta dry weight) was found to be identical for both C57Blk/6 mice and for mice with vascular smooth muscle-targeted expression of p22phox associated with strong increase in eNOS activity. Furthermore, the treatment of patients with cardiovascular diseases with carvedilol for 3 weeks increases up to 2 times the circulating NOHb concentration. These results demonstrate the important role of eNOS in the formation of circulating NOHb and suggest that NOHb can be used as a noninvasive marker of endothelial NO production in vivo.

• Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice.

  Authors: Karine Laude, Hua Cai, Bruno Fink, Nyssa Hoch, David S Weber, Louise McCann, Georg Kojda, Tohru Fukai, Harald H H W Schmidt, Sergey Dikalov, Santhini Ramasamy, Graciela Gamez, Kathy K Griendling, David G Harrison

  American journal of physiology. Heart and circulatory physiology. 02/2005; 288(1):H7-12.

  Protein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determineProtein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22(phox) expression would alter vascular ROS production and hemodynamics by targeting p22(phox) expression to smooth muscle in transgenic (Tg) mice. Aortas of Tg(p22smc) mice had increased p22(phox) and Nox1 protein levels and produced more superoxide and H(2)O(2). Surprisingly, endothelium-dependent relaxation and blood pressure in Tg(p22smc) mice were normal. Aortas of Tg(p22smc) mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tg(p22smc) mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tg(p22smc) mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tg(p22smc) mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H(2)O(2) production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.

• ESR techniques for the detection of nitric oxide in vivo and in tissues.

  Authors: Sergey Dikalov, Bruno Fink

  Methods in enzymology. 02/2005; 396:597-610.

  Plasma levels of nitrite/nitrate may not accurately reflect endothelial nitric oxide synthase (eNOS) function because of interference by dietary nitrates. Nitrosyl hemoglobin (HbNO), a metabolicPlasma levels of nitrite/nitrate may not accurately reflect endothelial nitric oxide synthase (eNOS) function because of interference by dietary nitrates. Nitrosyl hemoglobin (HbNO), a metabolic product of nitric oxide (NO*), may better correlate with bioavailable NO*, but it may depend on the activity of different NOS isoforms and may be affected by dietary nitrite/nitrate. This work examined the correlation between vascular endothelial NO* release and blood levels of HbNO. We measured HbNO in mouse blood using electron spin resonance (ESR) spectrometry, and we quantified vascular production of NO* using colloid Fe(DETC)2 and ESR. C57Blk/6 mice who were fed a high-nitrate diet had levels of plasma HbNO increased 10-fold, whereas those fed a low-nitrate diet had decreased HbNO levels from 0.58 +/- 0.02 to 0.48 +/- 0.01 microM. Therefore, a low-nitrate diet is essential when using HbNO as a marker of eNOS activity. Treatment with L-NAME and the eNOS-specific inhibitor L-NIO halved HbNO formation, which reflects the complete inhibition of NO* release by aorta endothelium. Treatment of mice with the selective inducible NOS (iNOS) inhibitor, 1400W, or the selective neuronal NOS (nNOS) inhibitor N-AANG did not alter either blood HbNO levels or vascular NO*. The relationship between HbNO and NO* production by the endothelium (0.23 microM HbNO to 5.27 microM/h of NO*/mg of dry weight aorta) was found to be identical for both C57Blk/6 mice and mice with vascular smooth muscle-targeted expression of p22phox associated with strong increase in eNOS activity. These results support the important role of eNOS in the formation of circulating HbNO, whereas iNOS and nNOS do not contribute to HbNO formation under normal conditions. These data suggest that HbNO can be used as a noninvasive marker of endothelial NO* production in vivo.

• Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay.

  Authors: Bruno Fink, Karine Laude, Louise McCann, Abdul Doughan, David G Harrison, Sergey Dikalov

  American journal of physiology. Cell physiology. 11/2004; 287(4):C895-902.

  Recently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in itsRecently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure. We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H(2)O(2), peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 microM menadione), and polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.

• C242T CYBA polymorphism of the NADPH oxidase is associated with reduced respiratory burst in human neutrophils.

  Authors: Keith E Wyche, Shaoshan S Wang, Kathy K Griendling, Sergey I Dikalov, Harland Austin, Swapna Rao, Bruno Fink, David G Harrison, A Maziar Zafari

  Hypertension. 06/2004; 43(6):1246-51.

  Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox-based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature.Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox-based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature. Some studies have identified reduced atherosclerosis in the presence of the C242T CYBA polymorphism, whereas others have not. Because vascular p22phox is identical to neutrophil p22phox, we studied the association between the C242T, A640G, and -930A/G CYBA polymorphisms and the quantity of superoxide produced from neutrophils isolated from healthy adults to determine if these polymorphisms had any functional impact on NADPH oxidase function. Neutrophils were isolated from 90 subjects by Percoll density gradient centrifugation. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping, as well as real-time PCR. The oxidative burst was stimulated with phorbol 12-myristate 13-acetate. Superoxide was quantified using the superoxide dismutase inhibitable oxidation of the spin probe hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, detected by electron paramagnetic resonance. Superoxide production was significantly affected by the C242T polymorphism, being 8.7+/-0.7, 7.9+/-0.6, and 5.9+/-1.2 micromol/L per minute per 10(6) neutrophils for the C242T CC, CT, and TT genotypes, respectively (P<0.05). In contrast, the A640G and the -930A/G polymorphisms did not alter the neutrophil respiratory burst. Phagocytic respiratory burst activity in homozygous individuals with the T allele of the C242T CYBA polymorphism is significantly lower than of wild-type carriers and heterozygous individuals. Because p22phox exists in both the neutrophil and vessel wall, vascular oxidative stress is likely diminished in individuals with this polymorphism.

• Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species.

  Authors: Andrey V Kozlov, Laszlo Szalay, Fraz Umar, Bruno Fink, Karl Kropik, Hans Nohl, Heinz Redl, Soheyl Bahrami

  Free radical biology & medicine. 07/2003; 34(12):1555-62.

  The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPRThe excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO(-) and O(2)(*)(-), as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.

• Association between vascular tolerance and platelet upregulation: comparison of nonintermittent administration of pentaerithrityltetranitrate and glyceryltrinitrate.

  Authors: Bruno Fink, Eberhard Bassenge

  Journal of cardiovascular pharmacology. 01/2003; 40(6):890-7.

  Enhanced formation of oxygen-derived radicals O plays a dominant role in the development of nitrate tolerance. In 18 healthy subjects, this study tested the effect of additional vitamin C (Vit-C)Enhanced formation of oxygen-derived radicals O plays a dominant role in the development of nitrate tolerance. In 18 healthy subjects, this study tested the effect of additional vitamin C (Vit-C) administration (1 g three times daily) on glyceryltrinitrate (GTN)-induced hemodynamic changes during 3 days of nonintermittent transdermal administration of GTN (0.4 mg/h) in comparison with administration of pentaerithrityltetranitrate (PETN, 40 mg three times daily, orally). GTN caused an immediate significant rise in arterial conductivity (a/b ratio of dicrotic pulse pressure, from 2.33 +/- 0.06 to 2.52 +/- 0.06). Within 2 days of GTN administration, the a/b ratio progressively decreased and reached pre-GTN control levels, documenting tolerance. However, the administration of GTN along with Vit-C or with PETN alone induced changes in the a/b ratio and in the orthostatic reaction, which were fully maintained for the period of treatment. This vascular tolerance seen after GTN treatment was paralleled by an upregulation of ex vivo platelet activity, which was evident from a rise in aggregation from 29.2 +/- 2.8% at control day to 85.4 +/- 8.5% at day 3, and additionally from thrombin-induced increases of intracellular Ca concentration from 494 +/- 60 nM at control day to 741 +/- 37 nM at day 3. This upregulation was not observed during PETN or GTN; with additional Vit-C administration. Administration of PETN or GTN, the latter supplemented by Vit-C, induced neither vascular tolerance nor the upregulation of washed platelet activity during nonintermittent administration, in contrast to GTN without Vit-C. This is explained by a diminished formation of reactive oxygen species when PETN or when GTN along with Vit-C is used.

• ESR Techniques for the Detection of Nitric Oxide In Vivo and in Tissues

  Authors: Sergey Dikalov, Bruno Fink

  Methods in Enzymology.

  Plasma levels of nitrite/nitrate may not accurately reflect endothelial nitric oxide synthase (eNOS) function because of interference by dietary nitrates. Nitrosyl hemoglobin (HbNO), a metabolicPlasma levels of nitrite/nitrate may not accurately reflect endothelial nitric oxide synthase (eNOS) function because of interference by dietary nitrates. Nitrosyl hemoglobin (HbNO), a metabolic product of nitric oxide (NO•), may better correlate with bioavailable NO•, but it may depend on the activity of different NOS isoforms and may be affected by dietary nitrite/nitrate. This work examined the correlation between vascular endothelial NO• release and blood levels of HbNO. We measured HbNO in mouse blood using electron spin resonance (ESR) spectrometry, and we quantified vascular production of NO• using colloid Fe(DETC)2 and ESR. C57Blk/6 mice who were fed a high‐nitrate diet had levels of plasma HbNO increased 10‐fold, whereas those fed a low‐nitrate diet had decreased HbNO levels from 0.58 ± 0.02 to 0.48 ± 0.01 μM. Therefore, a low‐nitrate diet is essential when using HbNO as a marker of eNOS activity. Treatment with l‐NAME and the eNOS‐specific inhibitor l‐NIO halved HbNO formation, which reflects the complete inhibition of NO• release by aorta endothelium. Treatment of mice with the selective inducible NOS (iNOS) inhibitor, 1400W, or the selective neuronal NOS (nNOS) inhibitor N‐AANG did not alter either blood HbNO levels or vascular NO•. The relationship between HbNO and NO• production by the endothelium (0.23 μM HbNO to 5.27 μM/h of NO•/mg of dry weight aorta) was found to be identical for both C57Blk/6 mice and mice with vascular smooth muscle–targeted expression of p22phox associated with strong increase in eNOS activity. These results support the important role of eNOS in the formation of circulating HbNO, whereas iNOS and nNOS do not contribute to HbNO formation under normal conditions. These data suggest that HbNO can be used as a noninvasive marker of endothelial NO• production in vivo.

• TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction

  Authors: Nithya Mariappan, Carrie M. Elks, Bruno Fink, Joseph Francis

  Free Radical Biology and Medicine.

  Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction.Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O2−) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O2− and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague–Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 μmol/kg bw, orally), and TNF+ Tempol (Temp; 300 μmol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O2− production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.