crossed the cell-specific knockout mice with low densitiy lipoproteinreceptor-deficientmice. These studies revealed an athero-protective roleof dendritic cell-expressed HIF1α in atherosclerosis.

doi:10.1016/j.vph.2011.08.178

P.13.4IL-1α-dependent sterile inflammation in atherosclerosis is celltype-dependent and driven by necrotic vascular smoothmuscle cellsYue Zheng, Melanie Humphry, Martin R. Bennett, Murray C.H. ClarkeDivision of Cardiovascular Medicine, University of Cambridge,Cambridge, UKE-mail address: mchc2@cam.ac.uk (M.C.H. Clarke)

Multiple cell types undergo apoptosis and necrosis withinadvanced human atherosclerotic plaques and levels are increased inunstable lesions. Cell death is associated with inflammation and bothreduce plaque stability, which may trigger myocardial infarction.However it is unclear whether it is the apoptotic cells themselves orthe downstream responses to them that mediate the associatedpathology. We find that VSMC apoptosis in mice with establishedplaques or hyperlipidaemia increases serum levels of the proathero-genic cytokines MCP-1, TNFα and IL-6, which associated withreduced levels of phagocytosis. In vitro, necrotic VSMC lysatesinduced release of IL-6 and MCP-1 from healthy VSMCs, and weidentify the active mediator as IL-1α both in vivo and in vitro. Incontrast, comparable amounts of IL-1α released from necroticmacrophages or T-cells failed to elicit an immune response. We find,in disagreement to the accepted literature, that IL-1α requiresprocessing by calpain for full biological activity, due to a loweraffinity of pro-IL-1α for the IL-1 receptor than mature-IL-1α. Indeednecrosis-induced IL-1α-dependent responses are highly cell type-dependent, and correlate with calpain cleavage of IL-1α duringnecrosis. Cells unable to cleave contain an intracellular factor thatstably binds IL-1α protecting it from processing and preventingcytokine activity. Thus, within the atherosclerotic plaque necroticVSMCs are unique in their ability to induce IL-1α-dependent sterileinflammation. Given the recent doubts over the role of IL-1β inatherosclerosis, but the clear dependency on the IL-1 receptor, thismay suggest that IL-1α is in fact the major IL-1 ligand that drivespathogenesis.

doi:10.1016/j.vph.2011.08.179

P.13.5Mast cell activation via complement factor C5a modulates veingraft remodeling and accelerated atherosclerosisMargreet R. de Vriesa,b, Anouk Wezelb,c, Abbey Schepersb,Johan Kuiperc, Ilze Botc, Paul H.A. Quaxa,baEinthoven Laboratory for Experimental VascularMedicine, TheNetherlandsbDepartment of Surgery, Leiden University Medical Center, Leiden,The NetherlandscDivision of Biopharmaceutics, Leiden/AmsterdamCenter for Drug Research,Gorlaeus Laboratories, Leiden University, Leiden, The NetherlandsE-mail address: margreet.devries@lumc.nl (M.R. de Vries)

Introduction: We previously showed that innate immunityfactors complement factor C3 and mast cells are involved inatherosclerosis and vein graft disease (VGD). The role of comple-ment-factor C5a in these processes is unknown. Mast cells expressC5a-receptors, and can be activated by complement-factor C5a. We

here studied the effect of C5a on mast cell activation in VGD andsubsequent 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 and3 d) and then increased from 7 to 28 d. C5a and C5aR levels (RNA andprotein) increased after surgery due to influx of inflammatory cells,then decreased and stabilized further on. In vitro C5a-induced mastcell-activation resulted in an increase of tryptase release by 13% andan increase of MCP-1 release by 9%.

To study the effect of mast cells on VGD, vein grafts were placed inmast cell deficient Kit(W− sh/W− sh) mice, in ApoE-KO mice withsystemic treatment with Cromolyn (a mast cell-stabilizer) or localapplication of dinitrofluorobenzene (DNP, a mast cell-activator) andcontrol mice (n=10/group). Mast cell deficiency and Cromolynstabilization resulted, after 28 d, in a decrease in vein graft-thickening(VGT) of 45% and 22% resp. DNP stimulation showed an increase ofVGT of 36%. Furthermore local C5a application resulted in an increaseof 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 directactivation of mast cells by C5a, mice were treated with C5a andCromolyn, VGT was decreased by 54% compared to C5a-treated mice,to the level of Cromolyn treated mice. This was accompanied by adecrease in foam cell content of 13%.

Conclusion: These data give a strong indication that the activationof mast cells via C5a plays an important role in VGD and can be apotential therapeutic target.

doi:10.1016/j.vph.2011.08.180

P.13.6Extracellular nucleotides enhance procoagulatory profile ofendothelial cells after TNFα stimulationJoanna Drozdowskaa, Barbara Zyżynska-Granicaa, Anna Paradowskaa,Robert Jarzynab, Dorota Maciejkoa,Elżbieta Kaczmarekc, Katarzyna KoziakaaMedical University of Warsaw, Warsaw, PolandbUniversity of Warsaw, Warsaw, PolandcHarvard Medical School, Boston, USAE-mail address: jdrozdowska@wum.edu.pl (J. Drozdowska)

Introduction: Tissue factor (TF) plays a key role not only inthrombin generation and fibrin formation, but also in inflammatoryresponses. It is well established that TF is upregulated in cytokine-stimulated endothelial cells (EC). It is also well know that cytokinesinduce nucleotide release from cells, and extracellular nucleotidesmay regulate thrombosis and fibrinolysis by modulating tissueplasminogen activator and plasminogen activator inhibitor-1 genera-tion and release. However, the role of extracellular nucleotides inregulation of TF expression and activity has not been established sofar. The aim of the study was to determine if extracellular nucleotidesaffect TF expression in EC.

Methods: Human umbilical vein EC were stimulated withnucleotides (ATP, ADP, and UTP), with and without TNFα. TF surfaceantigen was measured with flow cytometry. Total, i.e., surface andcytoplasmic, antigen content and activity of TF were measured withELISA and chromogenic substrate assay, respectively. The expressionof mRNA for TF was assessed by real-time PCR.

Results: Extracellular nucleotides had very weak stimulatoryeffect on TF mRNA and protein levels, compared to TNFα. However,nucleotides potentiated TNFα-induced effects in EC, such as TFexpression at the mRNA, protein and activity levels.

Conclusion: Extracellular nucleotides under proinflammatorycondition enhance the endothelial procoagulatory phenotype by

Abstracts372