- Email: [email protected]
Endothelial dysfunction after hypoxia-reoxygenation: Do in vitro models work?
e154
14th IVBM Abstracts
electrolyte balance. It was shown that half of the individual variability of the ACE plasma concentration is determined by an insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene. The relationship between insertion/deletion polymorphism in the ACE gene and number of cardiovascular diseases was observed however in case of abdominal aortic aneurysm contradictory results were obtained. The aim of the study was to determine if there is an association between the ACE genotype and susceptibility to abdominal aneurysm or Leriche syndrome. Methods: Based on the PCR analysis ACE genotypes (I/I; I/D and D/D) were determined in four selected groups: 115 patients with AAA, 132 patients with Leriche syndrome who underwent surgery, 144 healthy individuals from control group and 295 samples of random Polish population group. Genotypes were compared with demographic and clinical data of subjects and analyzed in relation to risk factors. Results: No significant differences in genotype and allele frequencies between AAA patients, patients with Leriche syndrome and control subjects were found. Conclusion: ACE gene polymorphism does not seem to be an important factor in development of abdominal aortic aneurysm or Leriche syndrome. doi:10.1016/j.vph.2006.08.398
B19.12 Clusterin functions as a chaperone in occlusive myocardial infarction Dong Hun Kim1, Jeong Hun Kim2, Jin Hyoung Kim3, Hyoung Ryeol Kim4, Kyung Hui Kim1, Kyu-Won Kim3, Young Suk Yu2 1
Department of Radiology, College of Medicine, Chosun University, Gwangju, Korea 2 Department of Ophthalmology, Seoul National University College, Seoul, Korea 3 Research Institute of Pharmaceutical Sciences, College of Pharma, Seoul, Korea 4 Department of Cardiac and Thoracic Surgery, Seoul National University, Seoul, Korea Purpose: To elucidate the function of clusterin in the cardiomyocyte damaged by a coronary artery occlusion. Methods: At 6 h and 2 weeks after coronary artery (left anterior descending branch) occlusion of the Sprague–Dawley rats, immunohistochemistry and western blotting for clusterin, and TUNEL were performed in the acute and chronic infarct hearts. MTT assay and DAPI staining in oxygen–glucose deprivation (OGD) with or without clusterin were performed in C2H9 cell line. PARP cleavage and the phosphorylation of Akt were also checked in OGD with or without clusterin. Results: Clusterin was primarily expressed in endocardium of normal and infarct heart. Its expression was increased around the acute and chronic infarct myocardium. Clusterin signifi-
cantly blocked apoptosis of C2H9 cells in OGD. The PARP cleavage in OGD was also blocked by clusterin. Conclusion: Clusterin plays a protective role in the cardiomyocyte damaged by a coronary artery occlusion. Clusterin may function as a cellular chaperone of the cardiomyocyte. doi:10.1016/j.vph.2006.08.399
B19.13 Endothelial dysfunction after hypoxia-reoxygenation: Do in vitro models work? Tamás Radovits1, Li-ni Lin1, Julia Zotkina1, Song-He Chen1, Siegfried Hagl1, Gábor Szabó1 1
Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany Hypoxia-reoxygenation causes tissue injury, mainly due to overproduction of free radicals and activation of leukocytes. Damage to the endothelium in these pathophysiological conditions is widely described, however there are only sporadic functional studies investigating in vitro vascular hypoxiareoxygenation. This methodological study compares results of in vivo and in vitro functional experiments. In canine and porcine in vivo experiments hearts were subjected to regional ischemia by clamping the left anterior descending coronary artery (LAD) followed by reperfusion. Blood flow was measured on the LAD with a perivascular ultrasonic flow probe and vascular resistance was calculated. Endothelium-dependent vasodilatation was assessed after single bolus intracoronary administration of acethylcholine (ACh), bradykinin (BK) and endothelium-independent vasodilatation after sodium nitroprusside (SNP).In in vitro experiments, isolated porcine coronary (LAD) and rat thoracic aortic rings were investigated in organ baths for isometric force measurements. Hypoxia (20, 45, 120 min) was induced in the chamber by gassing the Krebs–Henseleit solution with 95% N2–5%CO2. (pO2 < 30 mm Hg) During the subsequent reoxygenation (30, 45 min.), gassing was changed to 95%O2–5%CO2. The dose-dependent vasoresponse to BK, ACh and to SNP was investigated in precontracted rings under normoxic conditions and after hypoxia-reoxygenation. In additional experiments, porcine coronary arteries underwent ischaemia in vivo by clamping the LAD, then removed, reoxygenized, and vascular function was investigated in vitro under the same conditions in organ baths. Endothelial function (endothelium-dependent vasorelaxation) assessed by in vivo coronary blood flow measurements was significantly impaired after ischaemia-reperfusion. Although the typical hypoxic vasomotor response (transient contraction followed by slow relaxation) could be observed in the in vitro measurements, no impairment of endothelial function could be proven after hypoxia-reoxygenation in any groups of all investigated models. We conclude that endothelial injury occurring in vessel rings during in vitro hypoxia-reoxygenation is too slight (probably due to lack of activated leukocytes) and
14th IVBM Abstracts
cannot be demonstrated functionally. Therefore the experimental model of in vitro vascular hypoxia-reoxygenation is not suited for reliable investigations. doi:10.1016/j.vph.2006.08.400
B19.14 The effect of apolipoprotein j/clusterin on blood–retinal barrier breakdown of streptozotocin-induced diabetic rat Jeong Hun Kim1, Jin Hyoung Kim2, Yong Hae Son1, Kyu-Won Kim2, Young Suk Yu1 1
Department of Ophthalmology, Seoul National University, Seoul, Korea 2 College of Pharmacy, Seoul National University, Seoul, Korea Purpose: To determine the effect of clusterin on blood–retinal barrier breakdown of streptozotocin-induced diabetic rat. Methods: At 2, 4, 6, 8 days after intraperitoneal, streptozotocin injection of the Sprague–Dawley rats, immunohistochemistry and western blotting for clusterin, ZO-1, and ZO-2 were performed in the retina. MTT assay and DAPI staining in AGEtreated or hyperglycemic condition (in vitro diabetes-mimicking condition) with or without clusterin were performed in retinal endothelial cells. The restoration of blood–retinal barrier breakdown by treatment of clusterin was checked in retinal endothelial cells through western blotting for ZO-1 and ZO-2, and in retina through fluorescein angiography. Results: In the retina, clusterin expression was progressively enhanced with increasing time. Clusterin was prominently expressed in inner retina. In contrast, ZO-1 and ZO-2 were inversely decreased. In AGE-treated or hyperglycemic condition, clusterin protected retinal endothelial cells from apoptotic cell death. Clusterin rescued blood–retinal barrier from diabetes-induced breakdown. Conclusion: Diabetes affects the expression of clusterin in the retina. In diabetic retinopathy, clusterin may play a protective role in retinal endothelial cell injury and blood–retinal barrier breakdown.
2
Psychology Department, University of Auckland, Auckland, New Zealand Age-related cognitive decline has enormous social-economic impact. Using novel object recognition tests and elevated cross maze tests we examined the short-term memory and anxiety in young (4–6 months), middle aged (9–11 months) and the aged (18–20 months) Wistar rats. Our data show that a progressive decline in memory between middle aged and aged groups, with a reduced anxiety in middle aged rats. Using immunohistochemical staining, we also found that the loss of memory is correlated with the loss of neurons in the hippocampus, elevated GFAP positive astrocytes in the cerebral cortex and the hippocampus in aged rats, particularly those closely associated with blood vessels. Unexpectedly, we have found a transient increase in cell proliferation in sub-ventricular-zoon, with majority co-localized with neuroblasts. This transient up-regulation also found in multiple neuronal phenotypes of middle-aged rats in different brain regions, which co-relate to the reduced anxiety in middle aged rats. Glycine-2methyl-proline-glutamate (GMPE) is an analogue of the N-terminal tripeptide of insulin-like growth factor-1, glycone-proline-glutamate (GPE) [1,2]. GPE has been shown to be neuroprotective and up-regulates ChAT and NOS after ischemic brain injury [1]. Treatment with GMPE acutely promotes neurogenesis in both middle aged and aged group, and reduced GFAP positive atrocytes in the hippocampus and the cerebral cortex. Long-term treatment with GMPE also enhanced spatial memory in a dose dependent fashion. The study suggested the further enhancing neurogenesis may be important for improving memory in middle-aged brains and 2) inhibiting astrocytosis could be new approaches to ameliorating cognitive decline with aging. [1] Guan, J., Waldvogel, et al. Neuroscience, 89 (1999) 649– 659. [2] Harris, P.W.R., Brimble, M.A. et al. Tetrahedorn, in press. doi:10.1016/j.vph.2006.08.402
B19.16 Curcumin inhibits hypoxia-induced angiogenesis via downregulation of HIF-1
doi:10.1016/j.vph.2006.08.401
Su-Ryun Kim1, Soo-Kyung Bae2, Kyu-Won Kim2, Moon-Kyoung Bae1
B19.15
1
GMPE mediated aging associated neurogenesis and astrocytosis in aged rats Jian Guan1, Rong Zhang1, Cherry Chen1, Laura Jacobson1, Sam Mathai1, Dong Elliffe2, Peter Gluckman1, Di McCarthy2 1
The Liggins Institute, University of Auckland, Auckland, New Zealand
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College of Dentistry, Pusan National University, Pusan, Korea 2 College of Pharmacy, Seoul National University, Seoul, Korea Hypoxia inducible factor-1 (HIF-1) has a central role in cellular responses to hypoxia, including the transcriptional activation of a number of genes involved in angiogenesis in tumors. Here, we found that curcumin, a natural, biologically active compound isolated from the commonly used spice turmeric, significantly decreases hypoxia-induced HIF-1α
14th IVBM Abstracts
electrolyte balance. It was shown that half of the individual variability of the ACE plasma concentration is determined by an insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene. The relationship between insertion/deletion polymorphism in the ACE gene and number of cardiovascular diseases was observed however in case of abdominal aortic aneurysm contradictory results were obtained. The aim of the study was to determine if there is an association between the ACE genotype and susceptibility to abdominal aneurysm or Leriche syndrome. Methods: Based on the PCR analysis ACE genotypes (I/I; I/D and D/D) were determined in four selected groups: 115 patients with AAA, 132 patients with Leriche syndrome who underwent surgery, 144 healthy individuals from control group and 295 samples of random Polish population group. Genotypes were compared with demographic and clinical data of subjects and analyzed in relation to risk factors. Results: No significant differences in genotype and allele frequencies between AAA patients, patients with Leriche syndrome and control subjects were found. Conclusion: ACE gene polymorphism does not seem to be an important factor in development of abdominal aortic aneurysm or Leriche syndrome. doi:10.1016/j.vph.2006.08.398
B19.12 Clusterin functions as a chaperone in occlusive myocardial infarction Dong Hun Kim1, Jeong Hun Kim2, Jin Hyoung Kim3, Hyoung Ryeol Kim4, Kyung Hui Kim1, Kyu-Won Kim3, Young Suk Yu2 1
Department of Radiology, College of Medicine, Chosun University, Gwangju, Korea 2 Department of Ophthalmology, Seoul National University College, Seoul, Korea 3 Research Institute of Pharmaceutical Sciences, College of Pharma, Seoul, Korea 4 Department of Cardiac and Thoracic Surgery, Seoul National University, Seoul, Korea Purpose: To elucidate the function of clusterin in the cardiomyocyte damaged by a coronary artery occlusion. Methods: At 6 h and 2 weeks after coronary artery (left anterior descending branch) occlusion of the Sprague–Dawley rats, immunohistochemistry and western blotting for clusterin, and TUNEL were performed in the acute and chronic infarct hearts. MTT assay and DAPI staining in oxygen–glucose deprivation (OGD) with or without clusterin were performed in C2H9 cell line. PARP cleavage and the phosphorylation of Akt were also checked in OGD with or without clusterin. Results: Clusterin was primarily expressed in endocardium of normal and infarct heart. Its expression was increased around the acute and chronic infarct myocardium. Clusterin signifi-
cantly blocked apoptosis of C2H9 cells in OGD. The PARP cleavage in OGD was also blocked by clusterin. Conclusion: Clusterin plays a protective role in the cardiomyocyte damaged by a coronary artery occlusion. Clusterin may function as a cellular chaperone of the cardiomyocyte. doi:10.1016/j.vph.2006.08.399
B19.13 Endothelial dysfunction after hypoxia-reoxygenation: Do in vitro models work? Tamás Radovits1, Li-ni Lin1, Julia Zotkina1, Song-He Chen1, Siegfried Hagl1, Gábor Szabó1 1
Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany Hypoxia-reoxygenation causes tissue injury, mainly due to overproduction of free radicals and activation of leukocytes. Damage to the endothelium in these pathophysiological conditions is widely described, however there are only sporadic functional studies investigating in vitro vascular hypoxiareoxygenation. This methodological study compares results of in vivo and in vitro functional experiments. In canine and porcine in vivo experiments hearts were subjected to regional ischemia by clamping the left anterior descending coronary artery (LAD) followed by reperfusion. Blood flow was measured on the LAD with a perivascular ultrasonic flow probe and vascular resistance was calculated. Endothelium-dependent vasodilatation was assessed after single bolus intracoronary administration of acethylcholine (ACh), bradykinin (BK) and endothelium-independent vasodilatation after sodium nitroprusside (SNP).In in vitro experiments, isolated porcine coronary (LAD) and rat thoracic aortic rings were investigated in organ baths for isometric force measurements. Hypoxia (20, 45, 120 min) was induced in the chamber by gassing the Krebs–Henseleit solution with 95% N2–5%CO2. (pO2 < 30 mm Hg) During the subsequent reoxygenation (30, 45 min.), gassing was changed to 95%O2–5%CO2. The dose-dependent vasoresponse to BK, ACh and to SNP was investigated in precontracted rings under normoxic conditions and after hypoxia-reoxygenation. In additional experiments, porcine coronary arteries underwent ischaemia in vivo by clamping the LAD, then removed, reoxygenized, and vascular function was investigated in vitro under the same conditions in organ baths. Endothelial function (endothelium-dependent vasorelaxation) assessed by in vivo coronary blood flow measurements was significantly impaired after ischaemia-reperfusion. Although the typical hypoxic vasomotor response (transient contraction followed by slow relaxation) could be observed in the in vitro measurements, no impairment of endothelial function could be proven after hypoxia-reoxygenation in any groups of all investigated models. We conclude that endothelial injury occurring in vessel rings during in vitro hypoxia-reoxygenation is too slight (probably due to lack of activated leukocytes) and
14th IVBM Abstracts
cannot be demonstrated functionally. Therefore the experimental model of in vitro vascular hypoxia-reoxygenation is not suited for reliable investigations. doi:10.1016/j.vph.2006.08.400
B19.14 The effect of apolipoprotein j/clusterin on blood–retinal barrier breakdown of streptozotocin-induced diabetic rat Jeong Hun Kim1, Jin Hyoung Kim2, Yong Hae Son1, Kyu-Won Kim2, Young Suk Yu1 1
Department of Ophthalmology, Seoul National University, Seoul, Korea 2 College of Pharmacy, Seoul National University, Seoul, Korea Purpose: To determine the effect of clusterin on blood–retinal barrier breakdown of streptozotocin-induced diabetic rat. Methods: At 2, 4, 6, 8 days after intraperitoneal, streptozotocin injection of the Sprague–Dawley rats, immunohistochemistry and western blotting for clusterin, ZO-1, and ZO-2 were performed in the retina. MTT assay and DAPI staining in AGEtreated or hyperglycemic condition (in vitro diabetes-mimicking condition) with or without clusterin were performed in retinal endothelial cells. The restoration of blood–retinal barrier breakdown by treatment of clusterin was checked in retinal endothelial cells through western blotting for ZO-1 and ZO-2, and in retina through fluorescein angiography. Results: In the retina, clusterin expression was progressively enhanced with increasing time. Clusterin was prominently expressed in inner retina. In contrast, ZO-1 and ZO-2 were inversely decreased. In AGE-treated or hyperglycemic condition, clusterin protected retinal endothelial cells from apoptotic cell death. Clusterin rescued blood–retinal barrier from diabetes-induced breakdown. Conclusion: Diabetes affects the expression of clusterin in the retina. In diabetic retinopathy, clusterin may play a protective role in retinal endothelial cell injury and blood–retinal barrier breakdown.
2
Psychology Department, University of Auckland, Auckland, New Zealand Age-related cognitive decline has enormous social-economic impact. Using novel object recognition tests and elevated cross maze tests we examined the short-term memory and anxiety in young (4–6 months), middle aged (9–11 months) and the aged (18–20 months) Wistar rats. Our data show that a progressive decline in memory between middle aged and aged groups, with a reduced anxiety in middle aged rats. Using immunohistochemical staining, we also found that the loss of memory is correlated with the loss of neurons in the hippocampus, elevated GFAP positive astrocytes in the cerebral cortex and the hippocampus in aged rats, particularly those closely associated with blood vessels. Unexpectedly, we have found a transient increase in cell proliferation in sub-ventricular-zoon, with majority co-localized with neuroblasts. This transient up-regulation also found in multiple neuronal phenotypes of middle-aged rats in different brain regions, which co-relate to the reduced anxiety in middle aged rats. Glycine-2methyl-proline-glutamate (GMPE) is an analogue of the N-terminal tripeptide of insulin-like growth factor-1, glycone-proline-glutamate (GPE) [1,2]. GPE has been shown to be neuroprotective and up-regulates ChAT and NOS after ischemic brain injury [1]. Treatment with GMPE acutely promotes neurogenesis in both middle aged and aged group, and reduced GFAP positive atrocytes in the hippocampus and the cerebral cortex. Long-term treatment with GMPE also enhanced spatial memory in a dose dependent fashion. The study suggested the further enhancing neurogenesis may be important for improving memory in middle-aged brains and 2) inhibiting astrocytosis could be new approaches to ameliorating cognitive decline with aging. [1] Guan, J., Waldvogel, et al. Neuroscience, 89 (1999) 649– 659. [2] Harris, P.W.R., Brimble, M.A. et al. Tetrahedorn, in press. doi:10.1016/j.vph.2006.08.402
B19.16 Curcumin inhibits hypoxia-induced angiogenesis via downregulation of HIF-1
doi:10.1016/j.vph.2006.08.401
Su-Ryun Kim1, Soo-Kyung Bae2, Kyu-Won Kim2, Moon-Kyoung Bae1
B19.15
1
GMPE mediated aging associated neurogenesis and astrocytosis in aged rats Jian Guan1, Rong Zhang1, Cherry Chen1, Laura Jacobson1, Sam Mathai1, Dong Elliffe2, Peter Gluckman1, Di McCarthy2 1
The Liggins Institute, University of Auckland, Auckland, New Zealand
e155
College of Dentistry, Pusan National University, Pusan, Korea 2 College of Pharmacy, Seoul National University, Seoul, Korea Hypoxia inducible factor-1 (HIF-1) has a central role in cellular responses to hypoxia, including the transcriptional activation of a number of genes involved in angiogenesis in tumors. Here, we found that curcumin, a natural, biologically active compound isolated from the commonly used spice turmeric, significantly decreases hypoxia-induced HIF-1α