- Email: [email protected]
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Abstracts / Nitric Oxide 31 (2013) S13–S48
the perfusion signal reached the zero level. After 1–5 min of ischemia the cuff pressure was released. We determined intra-individual variability by measuring the same mouse on three consecutive days (n = 4) and inter-individual variability by comparing the coefficient of variation (CV) of 11 mice. The effect of acute and chronic inhibition of nitric oxide synthase (NOS) or cyclooxygenase (COX) on the hyperemic response was achieved by administration of L-NNA or indometacin i.p or for 6 days in drinking water ad libitum, respectively. Results: Application of nitroglycerin increased the perfusion of the hindlimb within approximately 100 s by 127% (191 ± 36 vs. 435 ± 23 perfusion units, PU), while epinephrine (209 ± 59 vs. 90 ± 18 PU:) resulted in a maximum decrease of 57% of the mean perfusion (n = 5). I.v. administration of adenosine elevated the perfusion by 41% (224 ± 34 vs. 316 ± 43 PU). Reactive hyperemia progressively increased after 1, 3 and 5 min ischemia with the maximal response achieved after 5 min ischemia. Time to maximum reperfusion and the ratio of maximum/baseline perfusion values (perfusion reserve) showed the smallest variation, while the area under the perfusion curve exhibited a CV of 40–50%. Generally, intra-individual variability was lower as compared to inter-individual variability. Chronic oral treatment with L-NNA significantly prolonged the time to maximal perfusion (36 ± 5 vs. 77 ± 18 s), while acute NOS inhibition (LNNA i.P) reduced the perfusion reserve (2.5 ± 0.1 vs. 1.8 ± 0.0). In addition, chronic inhibition of COX resulted in a 16 s longer reperfusion (48 ± 5 vs. 64 ± 5 s) but had no effect on the other parameters. Conclusion: Taken together, LDPI can be used as a valid and reproducible method for measuring vascular responses to physiological and pharmacological stimuli in the hindlimb of living mice. This may be a valuable experimental model that is similar to methodologies applied in humans enabling a translation of basic science findings. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.070
P69 Analysis of acute and chronic effects of ( )-epicatechin on NO bioavailability and organ redox state in the cardiovascular system in vivo
S43
and the effects analyzed after 1 h. Chronic effects were assessed after 5 days ( )-epicatechin administration of 2 and 10 mg/kg BW in drinking water. Vascular function was assessed by measuring reactive hyperemic response of the hindlimb by laser Doppler perfusion imaging (LDPI). Chronic inhibition of nitric oxide synthase (NOS) was achieved by administration of the specific inhibitor L-NNA in drinking water for 6 days. Circulating levels of ( )-epicatechin metabolites were determined with HPLC. In vitro effects of ( )epicatechin and its main plasma metabolites present in humans were analyzed in human umbilical vein endothelial cells (HUVECs). The H2O2 concentration in media was measured by a peroxide assay. The activation of Nrf2 by epicatechin was measured by immunohistochemistry and confocal microscopy of cells cultured on cover slips, Western blot analysis of nuclear and cytoplasmic extracts, as well as by ARE (antioxidant responsive element)-binding assay (TransAM). The expression of eNOS and of phase II genes was assessed in cells and organs by real time RT-PCR and Western blot analysis. Phosphorylation of eNOS was assessed by Western blot analysis. The concentration of reduced glutathione in mouse organs were assessed by the glutathione reductase recycling assay. Results: Acute administration of ( )-epicatechin dose-dependently increased the circulating levels of epicatechin metabolites along with improved vascular response to reactive hyperemia of the hindlimb. This was paralleled by an increase in eNOS phosphorylation in the aorta of the mice, indicating that ( )-epicatechin application induces NOS activation. Chronic treatment with ( )-epicatechin improved vascular response in a NOS-dependent fashion, and increased cGMP levels in aorta and plasma of the mice, demonstrating enhanced NO bioavailability. Interestingly, we observed a parallel increase in the expression of Nrf2-dependent phase II genes, including glutamate cysteine ligase (Gclc), and GSH levels in the heart and lung of the mice. In vitro we found that ( )-epicatechin induced the translocation of Nrf2 into the nucleus of the cells and the binding to the ARE. Moreover,( )-epicatechin and its main plasma metabolites increased expression of GCL (mRNA and protein), as well as eNOS expression and phosphorylation. Conclusions: Taken together, our findings demonstrate that administration of ( )-epicatechin leads to an increase of systemic NO bioavailability and vascular response in vivo and activates eNOS and Nrf2-dependent pathways. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.071
Miriam M. Cortese-Krott a, Thomas Krenz a, Ana Rodriguez-Matheos a,b, Friederike Oberle a, Kim Weber a, David Pullmann a, Magalie Haeberlein a, Sivatharsini Thasian-Sivarajah a, Jeremy Spencer b, Malte Kelm a, Christian Heiss a a University of Düsseldorf, Düsseldorf, Germany b University of Reading, Reading, United Kingdom Background and Hypothesis: Consumption of cocoa flavanols may have beneficial effects on cardiovascular health. ( )-Epicatechin, the major flavanol monomer present in cocoa, has been shown to positively affect endothelial function and a ( )-epicatechin-rich cocoa drink increased the circulating nitric oxide (NO) metabolites in humans. There is also evidence that flavanols and flavonoids may activate the expression and the activity of Nrf2-dependent phase II genes in cultured non-endothelial cells. We hypothesized that ( )-epicatechin may induce a systemic Nrf2-dependent antioxidant response, thereby contributing to maintain the redox state, NO bioavailability, and the functionality of the vasculature. Methods: In vivo effects of acute and chronic oral administration of ( )-epicatechin were analyzed in C57/Bl6 mice. ( )-Epicatechin was administrated at 1–10 mg/kg body weight (BW) intragastrically
P70 Molecular and physiological aspects connecting red cell eNOS, red blood cell deformability and decreased microvascular function in patients with essential hypertension Miriam M. Cortese-Krott, Roberto Sansone, Sivatharsini Thasian-Sivarajah, Maximilian Baaken, Maximilian Ziegler, David Vukjovic, Christian Heiss, Malte Kelm University of Düsseldorf, Düsseldorf, Germany Background: Arterial hypertension (HT) is a complex multifactorial condition associated with cardiovascular disease. Independent studies show that HT is associated with microcirculatory and hemorheological alterations, such as decreased red blood cell (RBC) deformability, as well as decreased endothelial function and nitric oxide (NO) bioavailability. Recently we found RBC carry an active endothelial nitric oxide synthase (eNOS), which is decreased in cardiovascular disease.Hypothesis: We hypothesized that a reduced
the perfusion signal reached the zero level. After 1–5 min of ischemia the cuff pressure was released. We determined intra-individual variability by measuring the same mouse on three consecutive days (n = 4) and inter-individual variability by comparing the coefficient of variation (CV) of 11 mice. The effect of acute and chronic inhibition of nitric oxide synthase (NOS) or cyclooxygenase (COX) on the hyperemic response was achieved by administration of L-NNA or indometacin i.p or for 6 days in drinking water ad libitum, respectively. Results: Application of nitroglycerin increased the perfusion of the hindlimb within approximately 100 s by 127% (191 ± 36 vs. 435 ± 23 perfusion units, PU), while epinephrine (209 ± 59 vs. 90 ± 18 PU:) resulted in a maximum decrease of 57% of the mean perfusion (n = 5). I.v. administration of adenosine elevated the perfusion by 41% (224 ± 34 vs. 316 ± 43 PU). Reactive hyperemia progressively increased after 1, 3 and 5 min ischemia with the maximal response achieved after 5 min ischemia. Time to maximum reperfusion and the ratio of maximum/baseline perfusion values (perfusion reserve) showed the smallest variation, while the area under the perfusion curve exhibited a CV of 40–50%. Generally, intra-individual variability was lower as compared to inter-individual variability. Chronic oral treatment with L-NNA significantly prolonged the time to maximal perfusion (36 ± 5 vs. 77 ± 18 s), while acute NOS inhibition (LNNA i.P) reduced the perfusion reserve (2.5 ± 0.1 vs. 1.8 ± 0.0). In addition, chronic inhibition of COX resulted in a 16 s longer reperfusion (48 ± 5 vs. 64 ± 5 s) but had no effect on the other parameters. Conclusion: Taken together, LDPI can be used as a valid and reproducible method for measuring vascular responses to physiological and pharmacological stimuli in the hindlimb of living mice. This may be a valuable experimental model that is similar to methodologies applied in humans enabling a translation of basic science findings. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.070
P69 Analysis of acute and chronic effects of ( )-epicatechin on NO bioavailability and organ redox state in the cardiovascular system in vivo
S43
and the effects analyzed after 1 h. Chronic effects were assessed after 5 days ( )-epicatechin administration of 2 and 10 mg/kg BW in drinking water. Vascular function was assessed by measuring reactive hyperemic response of the hindlimb by laser Doppler perfusion imaging (LDPI). Chronic inhibition of nitric oxide synthase (NOS) was achieved by administration of the specific inhibitor L-NNA in drinking water for 6 days. Circulating levels of ( )-epicatechin metabolites were determined with HPLC. In vitro effects of ( )epicatechin and its main plasma metabolites present in humans were analyzed in human umbilical vein endothelial cells (HUVECs). The H2O2 concentration in media was measured by a peroxide assay. The activation of Nrf2 by epicatechin was measured by immunohistochemistry and confocal microscopy of cells cultured on cover slips, Western blot analysis of nuclear and cytoplasmic extracts, as well as by ARE (antioxidant responsive element)-binding assay (TransAM). The expression of eNOS and of phase II genes was assessed in cells and organs by real time RT-PCR and Western blot analysis. Phosphorylation of eNOS was assessed by Western blot analysis. The concentration of reduced glutathione in mouse organs were assessed by the glutathione reductase recycling assay. Results: Acute administration of ( )-epicatechin dose-dependently increased the circulating levels of epicatechin metabolites along with improved vascular response to reactive hyperemia of the hindlimb. This was paralleled by an increase in eNOS phosphorylation in the aorta of the mice, indicating that ( )-epicatechin application induces NOS activation. Chronic treatment with ( )-epicatechin improved vascular response in a NOS-dependent fashion, and increased cGMP levels in aorta and plasma of the mice, demonstrating enhanced NO bioavailability. Interestingly, we observed a parallel increase in the expression of Nrf2-dependent phase II genes, including glutamate cysteine ligase (Gclc), and GSH levels in the heart and lung of the mice. In vitro we found that ( )-epicatechin induced the translocation of Nrf2 into the nucleus of the cells and the binding to the ARE. Moreover,( )-epicatechin and its main plasma metabolites increased expression of GCL (mRNA and protein), as well as eNOS expression and phosphorylation. Conclusions: Taken together, our findings demonstrate that administration of ( )-epicatechin leads to an increase of systemic NO bioavailability and vascular response in vivo and activates eNOS and Nrf2-dependent pathways. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.071
Miriam M. Cortese-Krott a, Thomas Krenz a, Ana Rodriguez-Matheos a,b, Friederike Oberle a, Kim Weber a, David Pullmann a, Magalie Haeberlein a, Sivatharsini Thasian-Sivarajah a, Jeremy Spencer b, Malte Kelm a, Christian Heiss a a University of Düsseldorf, Düsseldorf, Germany b University of Reading, Reading, United Kingdom Background and Hypothesis: Consumption of cocoa flavanols may have beneficial effects on cardiovascular health. ( )-Epicatechin, the major flavanol monomer present in cocoa, has been shown to positively affect endothelial function and a ( )-epicatechin-rich cocoa drink increased the circulating nitric oxide (NO) metabolites in humans. There is also evidence that flavanols and flavonoids may activate the expression and the activity of Nrf2-dependent phase II genes in cultured non-endothelial cells. We hypothesized that ( )-epicatechin may induce a systemic Nrf2-dependent antioxidant response, thereby contributing to maintain the redox state, NO bioavailability, and the functionality of the vasculature. Methods: In vivo effects of acute and chronic oral administration of ( )-epicatechin were analyzed in C57/Bl6 mice. ( )-Epicatechin was administrated at 1–10 mg/kg body weight (BW) intragastrically
P70 Molecular and physiological aspects connecting red cell eNOS, red blood cell deformability and decreased microvascular function in patients with essential hypertension Miriam M. Cortese-Krott, Roberto Sansone, Sivatharsini Thasian-Sivarajah, Maximilian Baaken, Maximilian Ziegler, David Vukjovic, Christian Heiss, Malte Kelm University of Düsseldorf, Düsseldorf, Germany Background: Arterial hypertension (HT) is a complex multifactorial condition associated with cardiovascular disease. Independent studies show that HT is associated with microcirculatory and hemorheological alterations, such as decreased red blood cell (RBC) deformability, as well as decreased endothelial function and nitric oxide (NO) bioavailability. Recently we found RBC carry an active endothelial nitric oxide synthase (eNOS), which is decreased in cardiovascular disease.Hypothesis: We hypothesized that a reduced