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ROK and NO contribute to vascular hyporeactivity in endotoxaemic rats
Abstracts / Nitric Oxide 27 (2012) S2–S50
Sport science and endothelial function P-15 Role of perivascular adipose tissue in endothelial dysfunction of adipose triglyceride lipase-deficient mice Karoline Dörffel a, Sarah Winkler a, Günter Hämmerle b, Rudolf Zechner b, Alois Lametschwandtner c, Bernd Mayer a, Astrid Schrammel-Gorren a a Department of Pharmacology and Toxicology, Karl-Franzens University, Graz, Austria, b Department of Molecular Biosciences, Karl-Franzens University, Graz, Austria, c Division of Zoology and Functional Anatomy, Vessel and Muscle Research Unit, University of Salzburg, Salzburg, Austria Perivascular adipose tissue (PVAT) is an important factor in vascular biology due to its ability to produce a variety of vasoactive substances. In addition, it is regarded as a source of proinflammatory mediators and reactive oxygen species (ROS). We have recently demonstrated that mice lacking adipose triglyceride lipase (ATGL) suffer from severe endothelial dysfunction. Since blood vessels of ATGL knockout ðATGLð= ÞÞ mice are covered by large amounts of PVAT, we investigated its potential contribution to the observed endothelial defect. Therefore, PVAT encompassing thoracic aortas of wildtype and ATGLð= Þ mice was isolated, and analyzed for expression of different adipokines, inflammation markers and sources of oxidative stress. Knockout of ATGL caused a 7-fold increase in PVAT wet weight. While adiponectin protein expression was not affected by ATGL deficiency, leptin levels were increased 3-fold. Adipose mRNA levels of the inflammation markers tumor necrosis factor a (TNFa and monocyte chemoattractant protein 1 (MCP-1) were 5fold higher in ATGL-deficient PVAT. In addition, NADPH oxidase isoform NOX2 was significantly upregulated at both mRNA and protein level. To distinguish between PVAT-mediated effects and those originating from the cardiac dysfunctional phenotype of the animals, we additionally analyzed PVAT isolated from ATGLð= Þ mice with cardiomyocyte-specific overexpression of ATGL (rescued cardiac phenotype). Interestingly, the effect of ATGL deficiency on TNFa and leptin expression was reversible, while increased adipose NOX2 and MCP-1 expression persisted. These data suggest that PVAT-derived inflammatory and oxidative stress might contribute to endothelial dysfunction in ATGL deficiency. The functional consequences of these observations are currently being investigated. http://dx.doi.org/10.1016/j.niox.2012.04.073
P-16 RhoA/ROK and NO contribute to vascular hyporeactivity in endotoxaemic rats Shiu-Jen Chen a,b, Mei-Hui Liao a, Chin-Chen Wu a a National Defence Medical Centre, Taipei, Taiwan, b Kang-Ning Junior College of Medical Care and Management, Taipei, Taiwan Hypotension and vascular hyporeactivity are significant characteristics of septic shock. The RhoA/Rho-kinase (RhoA/ROK) pathway is a major target for regulating calcium sensitivity of agonistinduced contraction. Here, we investigated the role of RhoA/ROK pathway in vascular hyporeactivity in endotoxaemic animals. Male Wistar rats were intravenously infused with E. coli lipopolysaccharide (LPS, 10 mg/kg) to induce endotoxaemia. Experimental animals were assigned to five groups: (i) 6 h after saline (Control); (ii) 1 h
S21
after LPS (LPS1h); (iii) 2 h after LPS (LPS2h); (iv) 4 h after LPS (LPS4h); (v) 6 h after LPS (LPS6h). LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Results showed that LPS induced a biphasic hypotension and sustained vascular hyporeactivity in vivo, and this hyporeactivity only occurred in late endotoxaemia ex vivo. Meanwhile, the aortic inducible nitric oxide (NO) synthase expression and the serum NO level were time-dependently increased after LPS. However, the aortic RhoA activity and myosin phosphatase targeting subunit 1 phosphorylation were increased in early endotoxaemia and this vascular reactivity was inhibited by ROK inhibitor, Y27632. In addition, the plasma bradykinin level was increased at 15 min and aortic endothelial NO synthase expression was increased 1 h after LPS. In conclusion, an increased RhoA activity may compensate vascular hyporeactivity in early endotoxaemia, whereas in late endotoxaemia, the large amount of NO produced causes inhibition of RhoA activity leading to vascular hyporeactivity in vivo and ex vivo. http://dx.doi.org/10.1016/j.niox.2012.04.074
P-17 Cytosolic expression of aldehyde dehydrogenase-2 is essential for nitroglycerin bioactivation by cultured cells Regina Neubauer, Matteo Beretta, Astrid Schrammel, Kurt Schmidt, Bernd Mayer Karl-Franzens-Universität, Graz, Austria We have recently shown that aldehyde dehydrogenase-2 (ALDH2) is mainly cytosolic in murine vascular tissue. Relaxation of ALDH2 knockout aortas to nitroglycerin (GTN) was restored by cytosolic but not by mitochondrial overexpression of ALDH2. To find out whether GTN bioactivation is determined by the endogenous distribution of ALDH2, we studied two types of cultured cells with strictly different ALDH2 localization for GTN-induced formation of 1,2-glycerol dinitrate (1,2-GDN) and accumulation of intracellular cGMP. Chloral hydrate (1 mM) and daidzin (0.1 mM) were used as ALDH inhibitors. Quantitative immunoblotting revealed that porcine aortic endothelial cells (PAECs) and rat lung fibroblasts (RFL-6 cells) expressed the protein predominantly in mitochondrial and cytosolic fractions, respectively. Interestingly, ALDH2-catalyzed GTN metabolism required addition of dithiothreitol (DTT), indicating that the cultured cells lack an essential reductant that is present in blood vessels. In the presence of 10 lM GTN and 2 mM DTT, the rates of 1,2-GDN formation were 0.5 ± 0.2 and 1.3 ± 0.4 pmol min1 mg1 in PAECs and RFL-6 cells, respectively. The maximal cGMP levels observed with 10 lM GTN in the presence of 2 mM DTT and 1 kU SOD/ml were 26 ± 4 and 87 ± 16 pmol/106 cell in PAECs and RFL-6 cells, corresponding to about 2- and 5-fold increases of basal levels, respectively. These results show that cytosolic expression of ALDH2 is essential for GTN bioactivation, indicating limited access of GTN to the mitochondrial matrix. Further work is needed to identify the endogenous reductant that was replaced by DTT in the present study. http://dx.doi.org/10.1016/j.niox.2012.04.075
P-18 Involvment of nitric oxide and MPTP in heart adaptation to physical training Vadym Sagach, Tatiana Shimanskaya, Yulia Goshovska Bogomolets Institute of Physiology, Kiev, Ukraine
Sport science and endothelial function P-15 Role of perivascular adipose tissue in endothelial dysfunction of adipose triglyceride lipase-deficient mice Karoline Dörffel a, Sarah Winkler a, Günter Hämmerle b, Rudolf Zechner b, Alois Lametschwandtner c, Bernd Mayer a, Astrid Schrammel-Gorren a a Department of Pharmacology and Toxicology, Karl-Franzens University, Graz, Austria, b Department of Molecular Biosciences, Karl-Franzens University, Graz, Austria, c Division of Zoology and Functional Anatomy, Vessel and Muscle Research Unit, University of Salzburg, Salzburg, Austria Perivascular adipose tissue (PVAT) is an important factor in vascular biology due to its ability to produce a variety of vasoactive substances. In addition, it is regarded as a source of proinflammatory mediators and reactive oxygen species (ROS). We have recently demonstrated that mice lacking adipose triglyceride lipase (ATGL) suffer from severe endothelial dysfunction. Since blood vessels of ATGL knockout ðATGLð= ÞÞ mice are covered by large amounts of PVAT, we investigated its potential contribution to the observed endothelial defect. Therefore, PVAT encompassing thoracic aortas of wildtype and ATGLð= Þ mice was isolated, and analyzed for expression of different adipokines, inflammation markers and sources of oxidative stress. Knockout of ATGL caused a 7-fold increase in PVAT wet weight. While adiponectin protein expression was not affected by ATGL deficiency, leptin levels were increased 3-fold. Adipose mRNA levels of the inflammation markers tumor necrosis factor a (TNFa and monocyte chemoattractant protein 1 (MCP-1) were 5fold higher in ATGL-deficient PVAT. In addition, NADPH oxidase isoform NOX2 was significantly upregulated at both mRNA and protein level. To distinguish between PVAT-mediated effects and those originating from the cardiac dysfunctional phenotype of the animals, we additionally analyzed PVAT isolated from ATGLð= Þ mice with cardiomyocyte-specific overexpression of ATGL (rescued cardiac phenotype). Interestingly, the effect of ATGL deficiency on TNFa and leptin expression was reversible, while increased adipose NOX2 and MCP-1 expression persisted. These data suggest that PVAT-derived inflammatory and oxidative stress might contribute to endothelial dysfunction in ATGL deficiency. The functional consequences of these observations are currently being investigated. http://dx.doi.org/10.1016/j.niox.2012.04.073
P-16 RhoA/ROK and NO contribute to vascular hyporeactivity in endotoxaemic rats Shiu-Jen Chen a,b, Mei-Hui Liao a, Chin-Chen Wu a a National Defence Medical Centre, Taipei, Taiwan, b Kang-Ning Junior College of Medical Care and Management, Taipei, Taiwan Hypotension and vascular hyporeactivity are significant characteristics of septic shock. The RhoA/Rho-kinase (RhoA/ROK) pathway is a major target for regulating calcium sensitivity of agonistinduced contraction. Here, we investigated the role of RhoA/ROK pathway in vascular hyporeactivity in endotoxaemic animals. Male Wistar rats were intravenously infused with E. coli lipopolysaccharide (LPS, 10 mg/kg) to induce endotoxaemia. Experimental animals were assigned to five groups: (i) 6 h after saline (Control); (ii) 1 h
S21
after LPS (LPS1h); (iii) 2 h after LPS (LPS2h); (iv) 4 h after LPS (LPS4h); (v) 6 h after LPS (LPS6h). LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Results showed that LPS induced a biphasic hypotension and sustained vascular hyporeactivity in vivo, and this hyporeactivity only occurred in late endotoxaemia ex vivo. Meanwhile, the aortic inducible nitric oxide (NO) synthase expression and the serum NO level were time-dependently increased after LPS. However, the aortic RhoA activity and myosin phosphatase targeting subunit 1 phosphorylation were increased in early endotoxaemia and this vascular reactivity was inhibited by ROK inhibitor, Y27632. In addition, the plasma bradykinin level was increased at 15 min and aortic endothelial NO synthase expression was increased 1 h after LPS. In conclusion, an increased RhoA activity may compensate vascular hyporeactivity in early endotoxaemia, whereas in late endotoxaemia, the large amount of NO produced causes inhibition of RhoA activity leading to vascular hyporeactivity in vivo and ex vivo. http://dx.doi.org/10.1016/j.niox.2012.04.074
P-17 Cytosolic expression of aldehyde dehydrogenase-2 is essential for nitroglycerin bioactivation by cultured cells Regina Neubauer, Matteo Beretta, Astrid Schrammel, Kurt Schmidt, Bernd Mayer Karl-Franzens-Universität, Graz, Austria We have recently shown that aldehyde dehydrogenase-2 (ALDH2) is mainly cytosolic in murine vascular tissue. Relaxation of ALDH2 knockout aortas to nitroglycerin (GTN) was restored by cytosolic but not by mitochondrial overexpression of ALDH2. To find out whether GTN bioactivation is determined by the endogenous distribution of ALDH2, we studied two types of cultured cells with strictly different ALDH2 localization for GTN-induced formation of 1,2-glycerol dinitrate (1,2-GDN) and accumulation of intracellular cGMP. Chloral hydrate (1 mM) and daidzin (0.1 mM) were used as ALDH inhibitors. Quantitative immunoblotting revealed that porcine aortic endothelial cells (PAECs) and rat lung fibroblasts (RFL-6 cells) expressed the protein predominantly in mitochondrial and cytosolic fractions, respectively. Interestingly, ALDH2-catalyzed GTN metabolism required addition of dithiothreitol (DTT), indicating that the cultured cells lack an essential reductant that is present in blood vessels. In the presence of 10 lM GTN and 2 mM DTT, the rates of 1,2-GDN formation were 0.5 ± 0.2 and 1.3 ± 0.4 pmol min1 mg1 in PAECs and RFL-6 cells, respectively. The maximal cGMP levels observed with 10 lM GTN in the presence of 2 mM DTT and 1 kU SOD/ml were 26 ± 4 and 87 ± 16 pmol/106 cell in PAECs and RFL-6 cells, corresponding to about 2- and 5-fold increases of basal levels, respectively. These results show that cytosolic expression of ALDH2 is essential for GTN bioactivation, indicating limited access of GTN to the mitochondrial matrix. Further work is needed to identify the endogenous reductant that was replaced by DTT in the present study. http://dx.doi.org/10.1016/j.niox.2012.04.075
P-18 Involvment of nitric oxide and MPTP in heart adaptation to physical training Vadym Sagach, Tatiana Shimanskaya, Yulia Goshovska Bogomolets Institute of Physiology, Kiev, Ukraine