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Mast cell activation via complement factor C5a modulates vein graft remodeling and accelerated atherosclerosis
372
Abstracts
crossed the cell-specific knockout mice with low densitiy lipoprotein receptor-deficient mice. These studies revealed an athero-protective role of dendritic cell-expressed HIF1α in atherosclerosis.
doi:10.1016/j.vph.2011.08.178
P.13.4 IL-1α-dependent sterile inflammation in atherosclerosis is cell type-dependent and driven by necrotic vascular smooth muscle cells Yue Zheng, Melanie Humphry, Martin R. Bennett, Murray C.H. Clarke Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK E-mail address: [email protected] (M.C.H. Clarke) Multiple cell types undergo apoptosis and necrosis within advanced human atherosclerotic plaques and levels are increased in unstable lesions. Cell death is associated with inflammation and both reduce plaque stability, which may trigger myocardial infarction. However it is unclear whether it is the apoptotic cells themselves or the downstream responses to them that mediate the associated pathology. We find that VSMC apoptosis in mice with established plaques or hyperlipidaemia increases serum levels of the proatherogenic cytokines MCP-1, TNFα and IL-6, which associated with reduced levels of phagocytosis. In vitro, necrotic VSMC lysates induced release of IL-6 and MCP-1 from healthy VSMCs, and we identify the active mediator as IL-1α both in vivo and in vitro. In contrast, comparable amounts of IL-1α released from necrotic macrophages or T-cells failed to elicit an immune response. We find, in disagreement to the accepted literature, that IL-1α requires processing by calpain for full biological activity, due to a lower affinity of pro-IL-1α for the IL-1 receptor than mature-IL-1α. Indeed necrosis-induced IL-1α-dependent responses are highly cell typedependent, and correlate with calpain cleavage of IL-1α during necrosis. Cells unable to cleave contain an intracellular factor that stably binds IL-1α protecting it from processing and preventing cytokine activity. Thus, within the atherosclerotic plaque necrotic VSMCs are unique in their ability to induce IL-1α-dependent sterile inflammation. Given the recent doubts over the role of IL-1β in atherosclerosis, but the clear dependency on the IL-1 receptor, this may suggest that IL-1α is in fact the major IL-1 ligand that drives pathogenesis. doi:10.1016/j.vph.2011.08.179
P.13.5 Mast cell activation via complement factor C5a modulates vein graft remodeling and accelerated atherosclerosis Margreet R. de Vriesa,b, Anouk Wezelb,c, Abbey Schepersb, Johan Kuiperc, Ilze Botc, Paul H.A. Quaxa,b a Einthoven Laboratory for Experimental Vascular Medicine, The Netherlands b Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands c Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands E-mail address: [email protected] (M.R. de Vries) Introduction: We previously showed that innate immunity factors complement factor C3 and mast cells are involved in atherosclerosis and vein graft disease (VGD). The role of complement-factor C5a in these processes is unknown. Mast cells express C5a-receptors, and can be activated by complement-factor C5a. We
here studied the effect of C5a on mast cell activation in VGD and subsequent accelerated atherosclerosis in ApoE-KO mice. Methods and results: In murine vein grafts (n = 3–4/time point) perivascular mast cell numbers decreased after surgery (6 h, 1 and 3 d) and then increased from 7 to 28 d. C5a and C5aR levels (RNA and protein) increased after surgery due to influx of inflammatory cells, then decreased and stabilized further on. In vitro C5a-induced mast cell-activation resulted in an increase of tryptase release by 13% and an increase of MCP-1 release by 9%. To study the effect of mast cells on VGD, vein grafts were placed in mast cell deficient Kit(W− sh/W− sh) mice, in ApoE-KO mice with systemic treatment with Cromolyn (a mast cell-stabilizer) or local application of dinitrofluorobenzene (DNP, a mast cell-activator) and control mice (n = 10/group). Mast cell deficiency and Cromolyn stabilization resulted, after 28 d, in a decrease in vein graft-thickening (VGT) of 45% and 22% resp. DNP stimulation showed an increase of VGT of 36%. Furthermore local C5a application resulted in an increase of 79% of VGT accompanied by an increase in perivascular mast cells. Systemic application of a C5aR-antagonist resulted in decreased VGT (40%), and a reduction in number of MC by 40%. To assess the direct activation of mast cells by C5a, mice were treated with C5a and Cromolyn, VGT was decreased by 54% compared to C5a-treated mice, to the level of Cromolyn treated mice. This was accompanied by a decrease in foam cell content of 13%. Conclusion: These data give a strong indication that the activation of mast cells via C5a plays an important role in VGD and can be a potential therapeutic target. doi:10.1016/j.vph.2011.08.180
P.13.6 Extracellular nucleotides enhance procoagulatory profile of endothelial cells after TNFα stimulation Joanna Drozdowskaa, Barbara Zyżynska-Granicaa, Anna Paradowskaa, Robert Jarzynab, Dorota Maciejkoa, Elżbieta Kaczmarekc, Katarzyna Koziaka a Medical University of Warsaw, Warsaw, Poland b University of Warsaw, Warsaw, Poland c Harvard Medical School, Boston, USA E-mail address: [email protected] (J. Drozdowska) Introduction: Tissue factor (TF) plays a key role not only in thrombin generation and fibrin formation, but also in inflammatory responses. It is well established that TF is upregulated in cytokinestimulated endothelial cells (EC). It is also well know that cytokines induce nucleotide release from cells, and extracellular nucleotides may regulate thrombosis and fibrinolysis by modulating tissue plasminogen activator and plasminogen activator inhibitor-1 generation and release. However, the role of extracellular nucleotides in regulation of TF expression and activity has not been established so far. The aim of the study was to determine if extracellular nucleotides affect TF expression in EC. Methods: Human umbilical vein EC were stimulated with nucleotides (ATP, ADP, and UTP), with and without TNFα. TF surface antigen was measured with flow cytometry. Total, i.e., surface and cytoplasmic, antigen content and activity of TF were measured with ELISA and chromogenic substrate assay, respectively. The expression of mRNA for TF was assessed by real-time PCR. Results: Extracellular nucleotides had very weak stimulatory effect on TF mRNA and protein levels, compared to TNFα. However, nucleotides potentiated TNFα-induced effects in EC, such as TF expression at the mRNA, protein and activity levels. Conclusion: Extracellular nucleotides under proinflammatory condition enhance the endothelial procoagulatory phenotype by
Abstracts
crossed the cell-specific knockout mice with low densitiy lipoprotein receptor-deficient mice. These studies revealed an athero-protective role of dendritic cell-expressed HIF1α in atherosclerosis.
doi:10.1016/j.vph.2011.08.178
P.13.4 IL-1α-dependent sterile inflammation in atherosclerosis is cell type-dependent and driven by necrotic vascular smooth muscle cells Yue Zheng, Melanie Humphry, Martin R. Bennett, Murray C.H. Clarke Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK E-mail address: [email protected] (M.C.H. Clarke) Multiple cell types undergo apoptosis and necrosis within advanced human atherosclerotic plaques and levels are increased in unstable lesions. Cell death is associated with inflammation and both reduce plaque stability, which may trigger myocardial infarction. However it is unclear whether it is the apoptotic cells themselves or the downstream responses to them that mediate the associated pathology. We find that VSMC apoptosis in mice with established plaques or hyperlipidaemia increases serum levels of the proatherogenic cytokines MCP-1, TNFα and IL-6, which associated with reduced levels of phagocytosis. In vitro, necrotic VSMC lysates induced release of IL-6 and MCP-1 from healthy VSMCs, and we identify the active mediator as IL-1α both in vivo and in vitro. In contrast, comparable amounts of IL-1α released from necrotic macrophages or T-cells failed to elicit an immune response. We find, in disagreement to the accepted literature, that IL-1α requires processing by calpain for full biological activity, due to a lower affinity of pro-IL-1α for the IL-1 receptor than mature-IL-1α. Indeed necrosis-induced IL-1α-dependent responses are highly cell typedependent, and correlate with calpain cleavage of IL-1α during necrosis. Cells unable to cleave contain an intracellular factor that stably binds IL-1α protecting it from processing and preventing cytokine activity. Thus, within the atherosclerotic plaque necrotic VSMCs are unique in their ability to induce IL-1α-dependent sterile inflammation. Given the recent doubts over the role of IL-1β in atherosclerosis, but the clear dependency on the IL-1 receptor, this may suggest that IL-1α is in fact the major IL-1 ligand that drives pathogenesis. doi:10.1016/j.vph.2011.08.179
P.13.5 Mast cell activation via complement factor C5a modulates vein graft remodeling and accelerated atherosclerosis Margreet R. de Vriesa,b, Anouk Wezelb,c, Abbey Schepersb, Johan Kuiperc, Ilze Botc, Paul H.A. Quaxa,b a Einthoven Laboratory for Experimental Vascular Medicine, The Netherlands b Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands c Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands E-mail address: [email protected] (M.R. de Vries) Introduction: We previously showed that innate immunity factors complement factor C3 and mast cells are involved in atherosclerosis and vein graft disease (VGD). The role of complement-factor C5a in these processes is unknown. Mast cells express C5a-receptors, and can be activated by complement-factor C5a. We
here studied the effect of C5a on mast cell activation in VGD and subsequent accelerated atherosclerosis in ApoE-KO mice. Methods and results: In murine vein grafts (n = 3–4/time point) perivascular mast cell numbers decreased after surgery (6 h, 1 and 3 d) and then increased from 7 to 28 d. C5a and C5aR levels (RNA and protein) increased after surgery due to influx of inflammatory cells, then decreased and stabilized further on. In vitro C5a-induced mast cell-activation resulted in an increase of tryptase release by 13% and an increase of MCP-1 release by 9%. To study the effect of mast cells on VGD, vein grafts were placed in mast cell deficient Kit(W− sh/W− sh) mice, in ApoE-KO mice with systemic treatment with Cromolyn (a mast cell-stabilizer) or local application of dinitrofluorobenzene (DNP, a mast cell-activator) and control mice (n = 10/group). Mast cell deficiency and Cromolyn stabilization resulted, after 28 d, in a decrease in vein graft-thickening (VGT) of 45% and 22% resp. DNP stimulation showed an increase of VGT of 36%. Furthermore local C5a application resulted in an increase of 79% of VGT accompanied by an increase in perivascular mast cells. Systemic application of a C5aR-antagonist resulted in decreased VGT (40%), and a reduction in number of MC by 40%. To assess the direct activation of mast cells by C5a, mice were treated with C5a and Cromolyn, VGT was decreased by 54% compared to C5a-treated mice, to the level of Cromolyn treated mice. This was accompanied by a decrease in foam cell content of 13%. Conclusion: These data give a strong indication that the activation of mast cells via C5a plays an important role in VGD and can be a potential therapeutic target. doi:10.1016/j.vph.2011.08.180
P.13.6 Extracellular nucleotides enhance procoagulatory profile of endothelial cells after TNFα stimulation Joanna Drozdowskaa, Barbara Zyżynska-Granicaa, Anna Paradowskaa, Robert Jarzynab, Dorota Maciejkoa, Elżbieta Kaczmarekc, Katarzyna Koziaka a Medical University of Warsaw, Warsaw, Poland b University of Warsaw, Warsaw, Poland c Harvard Medical School, Boston, USA E-mail address: [email protected] (J. Drozdowska) Introduction: Tissue factor (TF) plays a key role not only in thrombin generation and fibrin formation, but also in inflammatory responses. It is well established that TF is upregulated in cytokinestimulated endothelial cells (EC). It is also well know that cytokines induce nucleotide release from cells, and extracellular nucleotides may regulate thrombosis and fibrinolysis by modulating tissue plasminogen activator and plasminogen activator inhibitor-1 generation and release. However, the role of extracellular nucleotides in regulation of TF expression and activity has not been established so far. The aim of the study was to determine if extracellular nucleotides affect TF expression in EC. Methods: Human umbilical vein EC were stimulated with nucleotides (ATP, ADP, and UTP), with and without TNFα. TF surface antigen was measured with flow cytometry. Total, i.e., surface and cytoplasmic, antigen content and activity of TF were measured with ELISA and chromogenic substrate assay, respectively. The expression of mRNA for TF was assessed by real-time PCR. Results: Extracellular nucleotides had very weak stimulatory effect on TF mRNA and protein levels, compared to TNFα. However, nucleotides potentiated TNFα-induced effects in EC, such as TF expression at the mRNA, protein and activity levels. Conclusion: Extracellular nucleotides under proinflammatory condition enhance the endothelial procoagulatory phenotype by