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3WS15-5 Coagulation proteases in vascular biology
185
Wednesday October 1, 2003: Workshop 3WS15 FIBRINOLYSIS AND PROTEOLYSIS IN VASCULAR DISEASE 3WS15-1
Intracellular proteolysis controlling oxygen responsiveness
P. Maxwell. Imperial College London, London, United Kingdom Maintaining oxygen homeostasis represents a fundamental challenge to multicellular organisms such as ourselves. Many aspects of meeting this challenge centre on the Hypoxia Inducible Factor-1 (HIF-1) transcription control system. A HIF-1 complex consists of an alpha and a beta subunit, both of which are members of multiprotein families. HIF-1 activity is regulated by oxygen through the alpha subunit through controlled proteolytic destruction and transactivator recruitment. In both instances the molecular mechanism is based on the enzymatic modification of a single atom of oxygen to specific residues. The enzymes responsible for controlling proteolysis have been termed PHD 1, 2 and 3 (for prolyl hydroxylase domain) and belong to the extended family of 2-oxoglutarate dependent dioxygenases. Following hydroxylation of either of two conserved prolyl residues, HIF alpha is captured by the von Hippel Lindau tumor suppressor protein. This acts as the recognition component of a ubiquitin E3 ligase complex and leads to ubiquitination and destruction by the proteosome. A related enzyme, FIH-1 (for factor inhibiting HIF-1) regulates transactivation by HIF alpha. In this case, hydroxylation of a conserved asparaginyl residue near the C terminus prevents the interaction between CBP/P300 and HIF alpha. The hydroxylase enzymes require oxygen as a cosubstrate and non-heme ferrous iron is a cofactor. This explains how the reaction functions as an oxygen sensor, and also why iron chelators activate the HIF-1 system. Inhibition of the enzymes with low molecular weight compounds is feasible and provides a means to induce or enhance HIF-1 activation. This should activate cellular adaptation, and enhance angiogenesis in ischaemic tissues. 3WS15-2
Oxidized low density lipoproteins induce interaction between a novel response element in plasminogen activator inhibitor-1 promoter and a nuclear protein from vascular endothelial cells
G. Shen, R. Zhao, L. Lu, X. Ma, L. Hu. University of Manitoba, Diabetes Research Group, Winnipeg, Manitoba, Canada
Coagulation proteases in vascular biology
W. Ruf. Scripps Research Institute, La Jolla, CA, USA Endothelial cell biology in development, angiogenesis, and inflammation is regulated by vascular cell signaling. We are interested in the role of protease cofactors in signaling of serine proteases through a subclass of G-protein coupled receptors, the protease activated receptors (PARs). PAR signaling in the pro- and anticoagulant pathways is linked to the initiation of protease cascades by cell surface receptors that bind serine proteases. In the initiation of coagulation, tissue factor (TF) forms a transient TF-VIIa-Xa complex in which Xa activates PAR1 or PAR2. In the anticoagulant protein C (PC) pathway, the thrombin-thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR). EPCR functions as a required co-receptor for signaling of activated PC through PAR1. TF and EPCR direct and restrict PAR activation and contribute to signaling specificity, e.g. the cytoplasmic domain of TF serves as a docking site for the recruitment of adaptors that co-signal. TF cytoplasmic domain function is regulated by phosphorylation. With a phosphorylation-specific antibody to Ser258 , we demonstrated that the TF cytoplasmic domain is primarily not phosphorylated in endothelial cells. Phosphorylation required Golgi-transport, intact cholesterol-rich microdomains, and cell surface expression of TF. Palmitoylation of Cys245 within the TF cytoplasmic domain negatively regulated Ser258 phosphorylation, suggesting a complex control that depends on localization of TF to specific membrane domains. PAR-signaling is a relevant agonist pathway that triggers PKC-dependent TF cytoplasmic domain phosphorylation. Deletion of the TF cytoplasmic domain results in deregulated angiogenesis, confirming relevance of the co-signaling functions of protease receptors in vivo.
3WS16 RECENT TOPICS IN RESEARCH ON HYPERTENSION AND ATHEROSCLEROSIS 3WS16-1
Mechanical stress-related atherosclerosis and functional genomics
H. Rakugi, T. Ogihara. Osaka University Graduate School of Medicine, Japan Pulsatile stretch and shear stress in hypertension are reported to participate in atherosclerosis. Relation of mechanical stress and atherosclerosis has been investigated from the point of biochemical molecules, such as angiotensin, endothelin, cytokines, and oxidative stress. Recent progress in gene analysis has revealed that some specific genes relate to both hypertension and carotid atherosclerosis. Examples of candidates are angiotensinogen, angiotensin-converting enzyme (ACE), endothelial nitric oxide synthesis, and methylenetetrahydrofolate reductase (MTHFR). So far, there is no reports on polymorphisms with linkage disequilibrium which relates to all of mechanical stress response, hypertension and atherosclerosis. In the case of ACE whose I/D polymorphism associates the gene expression very well, the promoter region contains shear stress responsive element but no polymorphism in this sequence has been reported. Therefore, mechanical stress may modify ACE expression in addition to I/D polymorphism related regulation. Furthermore, a protein which is regulated by mechanical stress such as Rho kinase, may modify expression of other genes which contribute to both hypertension and atherosclerosis. Although this is the most probable hypothesis, it is important how functionally analyze the relations between gene polymorphism and development of atherosclerosis, because atherosclerosis is caused by multiple factors. Plasma homocysteine concentration for MTHFR gene and serum ACE activity for ACE gene are well reported functional genomics in this field. We further investigated association of ICAM-1 gene polymorphism to IL-8 and VCAM-1 for atherosclerosis. The functional genomics is expected to reveal novel mechanisms of atherosclerosis.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
WEDNESDAY
High levels of plasminogen activator inhibitor-1 (PAI-1) are associated with cardiovascular disease and diabetes. Low density lipoprotein (LDL) and its oxidized forms stimulated PAI-1 production in cultured vascular endothelial cells (EC). The present study examined the effects of LDL and oxidized form on DNA-protein interaction on the PAI-1 promoter. Treatment with native LDL (0.1 mg/ml) activated -1528/+55 bp of the PAI-1 promoter/luciferase reporter gene transiently transfected in human umbilical vein EC by two-fold. Copper ion-oxidized LDL (oxLDL) at the same concentration activated the PAI-1 promoter by 4-fold. LDL and its oxidized form, but not very low density lipoprotein (VLDL), activated a VLDL-responsive element(-672/-657 bp)free mutant PAI-1 promoter transfected in EC. Native and oxLDL activated -1528/+55 bp and -1197/+55 bp of the PAI-1 promoter but not -1105/+55 bp or -804/+55 bp of the PAI-1 promoter. Treatment with oxLDL evidently increased the binding of a nuclear protein to an oligonucleotide within the targeted region of the PAI-1 promoter in eletrophoretic mobility shift assay. The binding was blocked by excess unlabeled oligonucleotides with identical sequence to the probe. The results demonstrated that oxLDL may activate a novel responsive element within -1197/-1105 bp region of the PAI-1 promoter, which is distinct from the previously reported VLDL-responsive element, through the binding of a nuclear protein to the within the targeted region for the putative oxLDL-responsive element. The DNA-protein interactions may contribute to oxLDL-induced overproduction of PAI-1 in EC. Supported by CIHR, UM, MHRC, MMSF, HSCF and St. James Kiwanis Club.
3WS15-5
Wednesday October 1, 2003: Workshop 3WS15 FIBRINOLYSIS AND PROTEOLYSIS IN VASCULAR DISEASE 3WS15-1
Intracellular proteolysis controlling oxygen responsiveness
P. Maxwell. Imperial College London, London, United Kingdom Maintaining oxygen homeostasis represents a fundamental challenge to multicellular organisms such as ourselves. Many aspects of meeting this challenge centre on the Hypoxia Inducible Factor-1 (HIF-1) transcription control system. A HIF-1 complex consists of an alpha and a beta subunit, both of which are members of multiprotein families. HIF-1 activity is regulated by oxygen through the alpha subunit through controlled proteolytic destruction and transactivator recruitment. In both instances the molecular mechanism is based on the enzymatic modification of a single atom of oxygen to specific residues. The enzymes responsible for controlling proteolysis have been termed PHD 1, 2 and 3 (for prolyl hydroxylase domain) and belong to the extended family of 2-oxoglutarate dependent dioxygenases. Following hydroxylation of either of two conserved prolyl residues, HIF alpha is captured by the von Hippel Lindau tumor suppressor protein. This acts as the recognition component of a ubiquitin E3 ligase complex and leads to ubiquitination and destruction by the proteosome. A related enzyme, FIH-1 (for factor inhibiting HIF-1) regulates transactivation by HIF alpha. In this case, hydroxylation of a conserved asparaginyl residue near the C terminus prevents the interaction between CBP/P300 and HIF alpha. The hydroxylase enzymes require oxygen as a cosubstrate and non-heme ferrous iron is a cofactor. This explains how the reaction functions as an oxygen sensor, and also why iron chelators activate the HIF-1 system. Inhibition of the enzymes with low molecular weight compounds is feasible and provides a means to induce or enhance HIF-1 activation. This should activate cellular adaptation, and enhance angiogenesis in ischaemic tissues. 3WS15-2
Oxidized low density lipoproteins induce interaction between a novel response element in plasminogen activator inhibitor-1 promoter and a nuclear protein from vascular endothelial cells
G. Shen, R. Zhao, L. Lu, X. Ma, L. Hu. University of Manitoba, Diabetes Research Group, Winnipeg, Manitoba, Canada
Coagulation proteases in vascular biology
W. Ruf. Scripps Research Institute, La Jolla, CA, USA Endothelial cell biology in development, angiogenesis, and inflammation is regulated by vascular cell signaling. We are interested in the role of protease cofactors in signaling of serine proteases through a subclass of G-protein coupled receptors, the protease activated receptors (PARs). PAR signaling in the pro- and anticoagulant pathways is linked to the initiation of protease cascades by cell surface receptors that bind serine proteases. In the initiation of coagulation, tissue factor (TF) forms a transient TF-VIIa-Xa complex in which Xa activates PAR1 or PAR2. In the anticoagulant protein C (PC) pathway, the thrombin-thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR). EPCR functions as a required co-receptor for signaling of activated PC through PAR1. TF and EPCR direct and restrict PAR activation and contribute to signaling specificity, e.g. the cytoplasmic domain of TF serves as a docking site for the recruitment of adaptors that co-signal. TF cytoplasmic domain function is regulated by phosphorylation. With a phosphorylation-specific antibody to Ser258 , we demonstrated that the TF cytoplasmic domain is primarily not phosphorylated in endothelial cells. Phosphorylation required Golgi-transport, intact cholesterol-rich microdomains, and cell surface expression of TF. Palmitoylation of Cys245 within the TF cytoplasmic domain negatively regulated Ser258 phosphorylation, suggesting a complex control that depends on localization of TF to specific membrane domains. PAR-signaling is a relevant agonist pathway that triggers PKC-dependent TF cytoplasmic domain phosphorylation. Deletion of the TF cytoplasmic domain results in deregulated angiogenesis, confirming relevance of the co-signaling functions of protease receptors in vivo.
3WS16 RECENT TOPICS IN RESEARCH ON HYPERTENSION AND ATHEROSCLEROSIS 3WS16-1
Mechanical stress-related atherosclerosis and functional genomics
H. Rakugi, T. Ogihara. Osaka University Graduate School of Medicine, Japan Pulsatile stretch and shear stress in hypertension are reported to participate in atherosclerosis. Relation of mechanical stress and atherosclerosis has been investigated from the point of biochemical molecules, such as angiotensin, endothelin, cytokines, and oxidative stress. Recent progress in gene analysis has revealed that some specific genes relate to both hypertension and carotid atherosclerosis. Examples of candidates are angiotensinogen, angiotensin-converting enzyme (ACE), endothelial nitric oxide synthesis, and methylenetetrahydrofolate reductase (MTHFR). So far, there is no reports on polymorphisms with linkage disequilibrium which relates to all of mechanical stress response, hypertension and atherosclerosis. In the case of ACE whose I/D polymorphism associates the gene expression very well, the promoter region contains shear stress responsive element but no polymorphism in this sequence has been reported. Therefore, mechanical stress may modify ACE expression in addition to I/D polymorphism related regulation. Furthermore, a protein which is regulated by mechanical stress such as Rho kinase, may modify expression of other genes which contribute to both hypertension and atherosclerosis. Although this is the most probable hypothesis, it is important how functionally analyze the relations between gene polymorphism and development of atherosclerosis, because atherosclerosis is caused by multiple factors. Plasma homocysteine concentration for MTHFR gene and serum ACE activity for ACE gene are well reported functional genomics in this field. We further investigated association of ICAM-1 gene polymorphism to IL-8 and VCAM-1 for atherosclerosis. The functional genomics is expected to reveal novel mechanisms of atherosclerosis.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
WEDNESDAY
High levels of plasminogen activator inhibitor-1 (PAI-1) are associated with cardiovascular disease and diabetes. Low density lipoprotein (LDL) and its oxidized forms stimulated PAI-1 production in cultured vascular endothelial cells (EC). The present study examined the effects of LDL and oxidized form on DNA-protein interaction on the PAI-1 promoter. Treatment with native LDL (0.1 mg/ml) activated -1528/+55 bp of the PAI-1 promoter/luciferase reporter gene transiently transfected in human umbilical vein EC by two-fold. Copper ion-oxidized LDL (oxLDL) at the same concentration activated the PAI-1 promoter by 4-fold. LDL and its oxidized form, but not very low density lipoprotein (VLDL), activated a VLDL-responsive element(-672/-657 bp)free mutant PAI-1 promoter transfected in EC. Native and oxLDL activated -1528/+55 bp and -1197/+55 bp of the PAI-1 promoter but not -1105/+55 bp or -804/+55 bp of the PAI-1 promoter. Treatment with oxLDL evidently increased the binding of a nuclear protein to an oligonucleotide within the targeted region of the PAI-1 promoter in eletrophoretic mobility shift assay. The binding was blocked by excess unlabeled oligonucleotides with identical sequence to the probe. The results demonstrated that oxLDL may activate a novel responsive element within -1197/-1105 bp region of the PAI-1 promoter, which is distinct from the previously reported VLDL-responsive element, through the binding of a nuclear protein to the within the targeted region for the putative oxLDL-responsive element. The DNA-protein interactions may contribute to oxLDL-induced overproduction of PAI-1 in EC. Supported by CIHR, UM, MHRC, MMSF, HSCF and St. James Kiwanis Club.
3WS15-5