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ANGIOGENESIS EFFECTS OF CHINESE HERBAL SALVIA MILTIORRHIZA AFTER ACUTE MYOCARDIAL INFARCTION IN RATS
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Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
sharply as repair mechanisms for oxidative damage are overwhelmed. Our objectives were to observe the effects of x-irradiation on endothelial cells from different species. Primary cultures of human, porcine or bovine aortic endothelial cells were irradiated with 250 kVp x-rays with total doses ranging from 0.125 to 16 Gy. Cells were assayed for clonogenic survival. At doses above 0.25 Gy in bovine cells and above 1 Gy in porcine and human cells, cells from all three species showed a typical decline in clonogenic survival with increasing dose. However, at lower doses, all three species differed from the predicted response. Bovine cells were stimulated to proliferate above the predicted rates. In contrast, proliferation of human and porcine cells declined steeply at all doses, suggesting that there was little repair of x-ray damage at any dose. Thus, human endothelial cells showed hypersensitivity at doses used in medical imaging.These responses do not fit with existing models of radiation damage and indicate that endothelial cells undergo changes in protein expression, which are protective in bovine cells but damaging to human and porcine cells. Thus, the endothelial cell response to irradiation may be unique and has implications for the development of cardiovascular disease including atherosclerosis. Health Canada
P426 DOCOSAHEXAENOIC ACID PREVENTS STRESS-INDUCED APOPTOSIS IN HUMAN ENDOTHELIAL CELLS. Claudia A. Vosseler, Peter C. Weber, Wolfgang Erl. Institut fuer Prophylaxe und Epidemiologie der Kreislaufkrankheiten. Background: Dysfunction and injury of the endothelium, including apoptosis of endothelial cells, is implicated in the initiation of atherosclerosis. A variety of vascular risk factors such as lipid peroxidation products have been shown to induce apoptosis in endothelial cells in vitro. On the other hand docosahexaenoic acid (DHA), a n-3 fatty acid, has been found to be protective towards the development of atherosclerosis. We therefore investigated whether DHA can modulate apoptosis in human umbilical vein endothelial cells (HUVEC). Methods: Confluent HUVECs were treated with DHA (20 mM), arachidonic acid (AA, 20 mM) or vehicle control for 24 hours. Apoptosis was induced by the lipid peroxidation product 4-hydroxynonenal (HNE, 20 mM, up to 24 h). Cell viability was assessed by trypan blue exclusion and MTT assays. Annexin V-FITC labelling of cells was used to detect apoptosis and analysed by flow cytometry. Dead cells were excluded by propidium iodide staining. The generation of superoxide anions was detected by FACScan analysis with hydroethidine staining. Intracellular glutathione (GSH) content was measured photometrically and protein expression was assessed by Western blot analysis. Results: HNE caused a time- and concentration-dependent induction of apoptosis in HUVECs. Pre-treatment with DHA significantly reduced the number of HNE-induced apoptotic cells by about 30%. This protective effect was evident after 6 hours of apoptosis induction. In contrast, pretreatment with AA increased the number of apoptotic cells within 6 hours of HNE treatment by 30%. The measurement of superoxide anion production showed a time-dependent increase after AA treatment and vehicle control whereas DHA treatment abolished the induction of oxidative stress by HNE. GSH analysis showed that DHA significantly increased the intracellular GSH content of HUVECs and reduced the HNE-induced decrease of GSH. In contrast, incubation of HUVECs with AA alone or together with HNE led to a decrease of GSH. A possible explanation for the protective effect of DHA may be the activation of the transcription factor NF-kB and subsequent anti-apoptotic pathways. Conclusion: HNE induces oxidative stress and apoptosis in HUVECs. Pretreatment of HUVECs with the n-3 fatty acid DHA but not the n-6 fatty acid AA showed reduced apoptosis induction. In contrast, AA intensified apoptosis. The HNE-induced decrease of GSH was counteracted by DHA, while AA decreased GSH further intensifying HNE effects. These
results suggest a mechanism by which DHA may improve endothelial function under conditions of oxidative stress.
P427 INTRACELLULAR IRON CHELATION BY DESFERRIOXAMINE INHIBITS LIPOPOLYSACCHARIDE-INDUCED ENDOTHELIAL ACTIVATION IN VIVO. Wei-Jian Zhang, Balz Frei. Linus Pauling Institute, Oregon State University, Corvallis, OR, USA. Endothelial adhesion molecule expression and monocyte recruitment are initiating events in atherosclerosis thought to be caused, in part, by shifts in the intracellular redox environment. Since redox-active transition metal ions, such as iron and copper, play an essential role in free radical production and, thus, intracellular redox environment, we hypothesized that these metal ions may also be involved in endothelial activation. We have shown previously that the intracellular iron- and copper-chelators, desferrioxamine (DFO) and neocuproine, respectively, inhibit TNFa-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) in human aortic endothelial cells. Therefore, the overall objective of this study was to investigate whether metal chelators can act as anti-inflammatory agents and prevent lipopolysaccharide (LPS)-induced endothelial activation in vivo. Male C57BL/6N mice were divided into three groups: control (saline, i.p. injection); LPS (50 mg/animal, i.p.); and DFO plus LPS (DFO 100 mg/kg body weight, daily i.p. for 14 days before LPS challenge). We found that DFO strongly inhibited LPS-induced increases in plasma concentrations of soluble VCAM-1 (sVCAM-1), soluble ICAM-1 (sICAM-1), and MCP-1. For sVCAM-1, the data were as follows: control, 625 ± 53 ng/ ml; LPS, 1125 ± 34 ng/ml; and DFO plus LPS, 741 ± 50 ng/ml; for sICAM1: control, 24.9 ± 0.9 mg/ml; LPS, 40.1 ± 3.4 mg/ml; and DFO plus LPS, 31.2 ± 1.2 mg/ml; and for MCP-1: control, 0.03 ± 0.01 ng/ml; LPS, 41.0 ± 9.2 ng/ml; and DFO plus LPS, 14.2 ± 7.4 ng/ml ( P < .05 compared to LPS treatment without DFO; n = 5 8). DFO also strongly inhibited LPSinduced NF-kB and AP-1 activation in mouse aorta ( P < .05; n = 5). In conclusion, our studies indicate that intracellular iron chelation by DFO inhibits LPS-induced endothelial activation in vivo. Therefore, metal chelation may be a novel strategy to prevent and treat atherosclerosis and other inflammatory diseases. This work was supported by U.S. National Institutes of Health grants ES11542 (WJZ) and HL-60886 (BF), and a Pilot Project Grant 2002 (WJZ) from Linus Pauling Institute, Oregon State University.
P428 ANGIOGENESIS EFFECTS OF CHINESE HERBAL SALVIA MILTIORRHIZA AFTER ACUTE MYOCARDIAL INFARCTION IN RATS. Yi Zhun Zhu, Xinyan Ji, Yi-Chun Zhu, Benny K-H Tan. Department of Pharmacology, National University of Singapore, Singapore, Dept. of Physiology, Shanghai Medical University, China. In the present study, we investigated effects of Chinese herbal extract Salvia miltiorrhiza on angiogenesis after MI and their underlying mechanisms of the beneficial effects on reducing infarct size following an acute MI. Experiments were performed in Wistar rats weighing 250-300g. All rats were treated with Saline or Salvia miltiorrhiza (mixture extracted from the root of Salviae miltiorrhiza, 1 g/kg/day) or Ramipril (angiotensinconverting enzyme inhibitor, pure compound, 1 mg/kg/day) for 1 week before ligation of the left descending coronary artery to induce MI. The treatment was continued for another 2 weeks after MI. Morphological examination and antioxidant assays were performed after sacrificing the animals at the end of the treatment period. Both Ramipril (135 ± 8 mm2) and Dan Shen (129 ± 9 mm2) reduced infarct size (IS) after MI when compared these ISs in saline treated group (160 ± 9 mm2). The mortalities
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 in both Ramipril and Dan Shen treated groups were significantly lower then saline treated group. Additionally, Dan Shen was found to increase the capillary density after MI but without direct effects on lowing blood pressure like Ramipril. Gene expression of vascular endothelial growth factor (VEGF) were found to be increased only in the Salvia miltiorrhiza treated group. Our results demonstrate that Salvia miltiorrhiza has similar cardioprotective effects after MI in rats as that of Ramipril. Its unique effects on angiogenesis could be one of the important mechanisms in protecting myocardium post MI.
Proteases in Vascular Biology and Pathobiology P429 EFFECTS OF HYDROXYMETHYLGLUTARYL-COENZYME A REDUCTASE INHIBITORS ON COMPONENTS OF THE FIBRINOLYTIC SYSTEM AND ON THE INFLAMMATORY MEDIATOR MCP-1 IN HUMAN CARDIAC MYOCYTES IN VITRO. S. Demyanets, C. Kaun, S. Pfaffenberger, T. Weiss, G. Zorn, G. Maurer, R. Pacher, K. Huber, J. Wojta. Department of Internal Medicine II, University of Vienna, Austria, 3rd Medical Department, Wilhelminenspital, Austria. Background: Several studies have shown that statin therapy reduces not only the incidence of cardiovascular events but also decreases mortality in patients with high or ‘‘average’’ cholesterol levels. However recently a significant number of investigations have given evidence that beyond their lipid lowering capacity statins exert ‘‘pleiotropic’’ actions resulting in vasoprotection. Among other effects hydroxymethylglutaryl-coenzyme A reductase inhibitors or statins have been shown to modulate the fibrinolytic system and the expression of inflammatory mediators in various cell types. We studied whether human adult cardiac myocytes (HACM) also represent a cellular target for such effects. We investigated possible effects of statins on plasminogen activator inhibitor-1 (PAI-1) and PAI-2 and on the inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1). Methods: HACM were cultured from ventricular tissue obtained from explanted recipients’ hearts after heart transplantation. Such cells were treated with different statins at concentrations from 0.01 mM to 5 mM for up to 96 h. Toxic effects of the statins used were excluded by monitoring lactate dehydrogenase leakage from such treated cells. Specific mRNA levels were determined by RT-PCR. PAI-1, MCP-1 in supernatants and PAI-2 in cell lysates were measured by specific ELISAs. Results: Simvastatin, fluvastatin, atorvastatin and lovastatin at a concentration of 5 mM downregulated PAI-1 specific mRNA by 57%, 46%, 43%, 33% and PAI-2 specific mRNA by 77%, 70%, 85%, 68%, respectively, in HACM. Simvastatin, atorvastatin and fluvastatin also downregulated MCP1 specific mRNA in these cells by 82%, 48% and 38%, respectively. As determined by ELISAs similar results were obtained on the level of protein production in HACM. Conclusion: Statins downregulate the expression of PAI-1, PAI-2 and MCP-1 in human cardiac myocytes. If such an effect is also operative in vivo the use of statins in clinical practice may contribute to the downregulation of these proteins in patients resulting in an increase in the fibrinolytic capacity and in a reduction of inflammatory activation directly in ventricular tissue. Austrian Academic Exchange Service
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P430 RELEASE OF AN ENDOGENOUS INHIBITOR OF THE PROPROTEIN CONVERTASE FURIN IN ACTIVATED HUMAN PLATELETS. Julie Leblond, Simon Gaudreau, Francine Grondin, Marie-He´le`ne Laprise, Claire M. Dubois. Service d’Immunologie, De´partement de Pe´diatrie, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada. The principal role of platelets is hemostasis while pathological thrombosis has been attributed to an inadequate regulation of thrombin production and/ or platelet activation. Numerous proteins participating in various aspects of platelet activation and the coagulation process, such as pro-von Willebrand factor, the platelet alphaIIbbeta3 integrin, and pro-factor IX, have been shown to be processed at cleavage sites recognized by the proprotein convertase furin. Furin, the prototype of the mammalian family of proprotein convertases, is known to cleave precursor proteins at the basic consensus sequence Arg-X-Lys/Arg-Arg resulting in the production of biologically active proteins. Furin is stored in human platelets and released upon their activation. Furin inhibition, using a synthetic peptide mimicking its recognition site, blocked platelet aggregation and the activation of the integrin alphaIIbbeta3. This study identifies the molecular forms of furin present in resting and activated platelets. Following platelet activation there is release of an active soluble form of furin of ~86kDa. The decreased immunodetection of this form of furin in time corresponds with the increase in formation of an 180 kDa immunoreactive complex. This high molecular weight complex has been identified as being composed of furin and the serpin PI8, an inhibitor of furin as well as other serine proteases. Immunoprecipitation of the PI8-furin complex confirms the interaction between furin and PI8. Enzymatic assays demonstrated that the loss in furin activity correlates with the increase in detection of the PI8-furin complex. The addition of the peptide-based furin inhibitor, Dec-RVKR-CH2Cl, inhibited the formation of the PI8-furin complex, indicating the need for an unoccupied active site of furin for the formation of this complex. Ammonium hydroxide treatment of the complex leads to its dissociation along with the reappearance of uncomplexed furin, confirming that the nature of this complex is indeed characteristic of a typical SDS-stable serpin-protease complex. Purification of both PI8 complexed and uncomplexed furin by FPLC demonstrated a loss in furin activity following its association with PI8. This suggests the existence of a regulatory mechanism limiting the actions of furin following platelet activation. Our results demonstrate for the first time the presence of an endogenous inhibitor of furin in a physiological system. Such a furin-PI8 pathway provides new insights into control mechanisms for regulating platelet aggregation. CIHR Research Grant CIHR Doctoral Research Award
P431 HIGHLY EXPRESSED LYSOSOMAL CATHEPSINS IN HUMAN ATHEROMA AND THEIR ROLE IN MACROPHAGE APOPTOSIS TRIGGED BY OXIDIZED LIPIDS. Wei Li, Xi-Ming Yuan. Division of Pathology II, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden. Background and Objectives: It has been recently noticed that atherosclerotic lesions in both human and mice over-express several lysosomal proteases including cathepsins B, D, L, and S, which are potent elastases and may participate in the remodelling of extra cellular matrix associated with the plaque stability. However, the mechanisms responsible for the over-expression of lysosomal cathepsins and related atherogenic implications remain unknown. In the present study, we aimed to (I) evaluate the expression of lysosomal cathepsins B and L in human carotid atheroma and their relation to apoptosis, (II) to test if oxidized LDL and oxysterols may
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
sharply as repair mechanisms for oxidative damage are overwhelmed. Our objectives were to observe the effects of x-irradiation on endothelial cells from different species. Primary cultures of human, porcine or bovine aortic endothelial cells were irradiated with 250 kVp x-rays with total doses ranging from 0.125 to 16 Gy. Cells were assayed for clonogenic survival. At doses above 0.25 Gy in bovine cells and above 1 Gy in porcine and human cells, cells from all three species showed a typical decline in clonogenic survival with increasing dose. However, at lower doses, all three species differed from the predicted response. Bovine cells were stimulated to proliferate above the predicted rates. In contrast, proliferation of human and porcine cells declined steeply at all doses, suggesting that there was little repair of x-ray damage at any dose. Thus, human endothelial cells showed hypersensitivity at doses used in medical imaging.These responses do not fit with existing models of radiation damage and indicate that endothelial cells undergo changes in protein expression, which are protective in bovine cells but damaging to human and porcine cells. Thus, the endothelial cell response to irradiation may be unique and has implications for the development of cardiovascular disease including atherosclerosis. Health Canada
P426 DOCOSAHEXAENOIC ACID PREVENTS STRESS-INDUCED APOPTOSIS IN HUMAN ENDOTHELIAL CELLS. Claudia A. Vosseler, Peter C. Weber, Wolfgang Erl. Institut fuer Prophylaxe und Epidemiologie der Kreislaufkrankheiten. Background: Dysfunction and injury of the endothelium, including apoptosis of endothelial cells, is implicated in the initiation of atherosclerosis. A variety of vascular risk factors such as lipid peroxidation products have been shown to induce apoptosis in endothelial cells in vitro. On the other hand docosahexaenoic acid (DHA), a n-3 fatty acid, has been found to be protective towards the development of atherosclerosis. We therefore investigated whether DHA can modulate apoptosis in human umbilical vein endothelial cells (HUVEC). Methods: Confluent HUVECs were treated with DHA (20 mM), arachidonic acid (AA, 20 mM) or vehicle control for 24 hours. Apoptosis was induced by the lipid peroxidation product 4-hydroxynonenal (HNE, 20 mM, up to 24 h). Cell viability was assessed by trypan blue exclusion and MTT assays. Annexin V-FITC labelling of cells was used to detect apoptosis and analysed by flow cytometry. Dead cells were excluded by propidium iodide staining. The generation of superoxide anions was detected by FACScan analysis with hydroethidine staining. Intracellular glutathione (GSH) content was measured photometrically and protein expression was assessed by Western blot analysis. Results: HNE caused a time- and concentration-dependent induction of apoptosis in HUVECs. Pre-treatment with DHA significantly reduced the number of HNE-induced apoptotic cells by about 30%. This protective effect was evident after 6 hours of apoptosis induction. In contrast, pretreatment with AA increased the number of apoptotic cells within 6 hours of HNE treatment by 30%. The measurement of superoxide anion production showed a time-dependent increase after AA treatment and vehicle control whereas DHA treatment abolished the induction of oxidative stress by HNE. GSH analysis showed that DHA significantly increased the intracellular GSH content of HUVECs and reduced the HNE-induced decrease of GSH. In contrast, incubation of HUVECs with AA alone or together with HNE led to a decrease of GSH. A possible explanation for the protective effect of DHA may be the activation of the transcription factor NF-kB and subsequent anti-apoptotic pathways. Conclusion: HNE induces oxidative stress and apoptosis in HUVECs. Pretreatment of HUVECs with the n-3 fatty acid DHA but not the n-6 fatty acid AA showed reduced apoptosis induction. In contrast, AA intensified apoptosis. The HNE-induced decrease of GSH was counteracted by DHA, while AA decreased GSH further intensifying HNE effects. These
results suggest a mechanism by which DHA may improve endothelial function under conditions of oxidative stress.
P427 INTRACELLULAR IRON CHELATION BY DESFERRIOXAMINE INHIBITS LIPOPOLYSACCHARIDE-INDUCED ENDOTHELIAL ACTIVATION IN VIVO. Wei-Jian Zhang, Balz Frei. Linus Pauling Institute, Oregon State University, Corvallis, OR, USA. Endothelial adhesion molecule expression and monocyte recruitment are initiating events in atherosclerosis thought to be caused, in part, by shifts in the intracellular redox environment. Since redox-active transition metal ions, such as iron and copper, play an essential role in free radical production and, thus, intracellular redox environment, we hypothesized that these metal ions may also be involved in endothelial activation. We have shown previously that the intracellular iron- and copper-chelators, desferrioxamine (DFO) and neocuproine, respectively, inhibit TNFa-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) in human aortic endothelial cells. Therefore, the overall objective of this study was to investigate whether metal chelators can act as anti-inflammatory agents and prevent lipopolysaccharide (LPS)-induced endothelial activation in vivo. Male C57BL/6N mice were divided into three groups: control (saline, i.p. injection); LPS (50 mg/animal, i.p.); and DFO plus LPS (DFO 100 mg/kg body weight, daily i.p. for 14 days before LPS challenge). We found that DFO strongly inhibited LPS-induced increases in plasma concentrations of soluble VCAM-1 (sVCAM-1), soluble ICAM-1 (sICAM-1), and MCP-1. For sVCAM-1, the data were as follows: control, 625 ± 53 ng/ ml; LPS, 1125 ± 34 ng/ml; and DFO plus LPS, 741 ± 50 ng/ml; for sICAM1: control, 24.9 ± 0.9 mg/ml; LPS, 40.1 ± 3.4 mg/ml; and DFO plus LPS, 31.2 ± 1.2 mg/ml; and for MCP-1: control, 0.03 ± 0.01 ng/ml; LPS, 41.0 ± 9.2 ng/ml; and DFO plus LPS, 14.2 ± 7.4 ng/ml ( P < .05 compared to LPS treatment without DFO; n = 5 8). DFO also strongly inhibited LPSinduced NF-kB and AP-1 activation in mouse aorta ( P < .05; n = 5). In conclusion, our studies indicate that intracellular iron chelation by DFO inhibits LPS-induced endothelial activation in vivo. Therefore, metal chelation may be a novel strategy to prevent and treat atherosclerosis and other inflammatory diseases. This work was supported by U.S. National Institutes of Health grants ES11542 (WJZ) and HL-60886 (BF), and a Pilot Project Grant 2002 (WJZ) from Linus Pauling Institute, Oregon State University.
P428 ANGIOGENESIS EFFECTS OF CHINESE HERBAL SALVIA MILTIORRHIZA AFTER ACUTE MYOCARDIAL INFARCTION IN RATS. Yi Zhun Zhu, Xinyan Ji, Yi-Chun Zhu, Benny K-H Tan. Department of Pharmacology, National University of Singapore, Singapore, Dept. of Physiology, Shanghai Medical University, China. In the present study, we investigated effects of Chinese herbal extract Salvia miltiorrhiza on angiogenesis after MI and their underlying mechanisms of the beneficial effects on reducing infarct size following an acute MI. Experiments were performed in Wistar rats weighing 250-300g. All rats were treated with Saline or Salvia miltiorrhiza (mixture extracted from the root of Salviae miltiorrhiza, 1 g/kg/day) or Ramipril (angiotensinconverting enzyme inhibitor, pure compound, 1 mg/kg/day) for 1 week before ligation of the left descending coronary artery to induce MI. The treatment was continued for another 2 weeks after MI. Morphological examination and antioxidant assays were performed after sacrificing the animals at the end of the treatment period. Both Ramipril (135 ± 8 mm2) and Dan Shen (129 ± 9 mm2) reduced infarct size (IS) after MI when compared these ISs in saline treated group (160 ± 9 mm2). The mortalities
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 in both Ramipril and Dan Shen treated groups were significantly lower then saline treated group. Additionally, Dan Shen was found to increase the capillary density after MI but without direct effects on lowing blood pressure like Ramipril. Gene expression of vascular endothelial growth factor (VEGF) were found to be increased only in the Salvia miltiorrhiza treated group. Our results demonstrate that Salvia miltiorrhiza has similar cardioprotective effects after MI in rats as that of Ramipril. Its unique effects on angiogenesis could be one of the important mechanisms in protecting myocardium post MI.
Proteases in Vascular Biology and Pathobiology P429 EFFECTS OF HYDROXYMETHYLGLUTARYL-COENZYME A REDUCTASE INHIBITORS ON COMPONENTS OF THE FIBRINOLYTIC SYSTEM AND ON THE INFLAMMATORY MEDIATOR MCP-1 IN HUMAN CARDIAC MYOCYTES IN VITRO. S. Demyanets, C. Kaun, S. Pfaffenberger, T. Weiss, G. Zorn, G. Maurer, R. Pacher, K. Huber, J. Wojta. Department of Internal Medicine II, University of Vienna, Austria, 3rd Medical Department, Wilhelminenspital, Austria. Background: Several studies have shown that statin therapy reduces not only the incidence of cardiovascular events but also decreases mortality in patients with high or ‘‘average’’ cholesterol levels. However recently a significant number of investigations have given evidence that beyond their lipid lowering capacity statins exert ‘‘pleiotropic’’ actions resulting in vasoprotection. Among other effects hydroxymethylglutaryl-coenzyme A reductase inhibitors or statins have been shown to modulate the fibrinolytic system and the expression of inflammatory mediators in various cell types. We studied whether human adult cardiac myocytes (HACM) also represent a cellular target for such effects. We investigated possible effects of statins on plasminogen activator inhibitor-1 (PAI-1) and PAI-2 and on the inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1). Methods: HACM were cultured from ventricular tissue obtained from explanted recipients’ hearts after heart transplantation. Such cells were treated with different statins at concentrations from 0.01 mM to 5 mM for up to 96 h. Toxic effects of the statins used were excluded by monitoring lactate dehydrogenase leakage from such treated cells. Specific mRNA levels were determined by RT-PCR. PAI-1, MCP-1 in supernatants and PAI-2 in cell lysates were measured by specific ELISAs. Results: Simvastatin, fluvastatin, atorvastatin and lovastatin at a concentration of 5 mM downregulated PAI-1 specific mRNA by 57%, 46%, 43%, 33% and PAI-2 specific mRNA by 77%, 70%, 85%, 68%, respectively, in HACM. Simvastatin, atorvastatin and fluvastatin also downregulated MCP1 specific mRNA in these cells by 82%, 48% and 38%, respectively. As determined by ELISAs similar results were obtained on the level of protein production in HACM. Conclusion: Statins downregulate the expression of PAI-1, PAI-2 and MCP-1 in human cardiac myocytes. If such an effect is also operative in vivo the use of statins in clinical practice may contribute to the downregulation of these proteins in patients resulting in an increase in the fibrinolytic capacity and in a reduction of inflammatory activation directly in ventricular tissue. Austrian Academic Exchange Service
S153
P430 RELEASE OF AN ENDOGENOUS INHIBITOR OF THE PROPROTEIN CONVERTASE FURIN IN ACTIVATED HUMAN PLATELETS. Julie Leblond, Simon Gaudreau, Francine Grondin, Marie-He´le`ne Laprise, Claire M. Dubois. Service d’Immunologie, De´partement de Pe´diatrie, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada. The principal role of platelets is hemostasis while pathological thrombosis has been attributed to an inadequate regulation of thrombin production and/ or platelet activation. Numerous proteins participating in various aspects of platelet activation and the coagulation process, such as pro-von Willebrand factor, the platelet alphaIIbbeta3 integrin, and pro-factor IX, have been shown to be processed at cleavage sites recognized by the proprotein convertase furin. Furin, the prototype of the mammalian family of proprotein convertases, is known to cleave precursor proteins at the basic consensus sequence Arg-X-Lys/Arg-Arg resulting in the production of biologically active proteins. Furin is stored in human platelets and released upon their activation. Furin inhibition, using a synthetic peptide mimicking its recognition site, blocked platelet aggregation and the activation of the integrin alphaIIbbeta3. This study identifies the molecular forms of furin present in resting and activated platelets. Following platelet activation there is release of an active soluble form of furin of ~86kDa. The decreased immunodetection of this form of furin in time corresponds with the increase in formation of an 180 kDa immunoreactive complex. This high molecular weight complex has been identified as being composed of furin and the serpin PI8, an inhibitor of furin as well as other serine proteases. Immunoprecipitation of the PI8-furin complex confirms the interaction between furin and PI8. Enzymatic assays demonstrated that the loss in furin activity correlates with the increase in detection of the PI8-furin complex. The addition of the peptide-based furin inhibitor, Dec-RVKR-CH2Cl, inhibited the formation of the PI8-furin complex, indicating the need for an unoccupied active site of furin for the formation of this complex. Ammonium hydroxide treatment of the complex leads to its dissociation along with the reappearance of uncomplexed furin, confirming that the nature of this complex is indeed characteristic of a typical SDS-stable serpin-protease complex. Purification of both PI8 complexed and uncomplexed furin by FPLC demonstrated a loss in furin activity following its association with PI8. This suggests the existence of a regulatory mechanism limiting the actions of furin following platelet activation. Our results demonstrate for the first time the presence of an endogenous inhibitor of furin in a physiological system. Such a furin-PI8 pathway provides new insights into control mechanisms for regulating platelet aggregation. CIHR Research Grant CIHR Doctoral Research Award
P431 HIGHLY EXPRESSED LYSOSOMAL CATHEPSINS IN HUMAN ATHEROMA AND THEIR ROLE IN MACROPHAGE APOPTOSIS TRIGGED BY OXIDIZED LIPIDS. Wei Li, Xi-Ming Yuan. Division of Pathology II, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden. Background and Objectives: It has been recently noticed that atherosclerotic lesions in both human and mice over-express several lysosomal proteases including cathepsins B, D, L, and S, which are potent elastases and may participate in the remodelling of extra cellular matrix associated with the plaque stability. However, the mechanisms responsible for the over-expression of lysosomal cathepsins and related atherogenic implications remain unknown. In the present study, we aimed to (I) evaluate the expression of lysosomal cathepsins B and L in human carotid atheroma and their relation to apoptosis, (II) to test if oxidized LDL and oxysterols may