Background: Circulating microparticles play an important role in cardiovascular diseases, as they mediate pro-inflammatory and pro-thrombotic activities [76]. A majority of circulating MPs is platelet-derived and their activities are mediated by both tissue factor (TF)-positive MPs, but also TF-negative MPs; e.g. by modulating the thrombogenic state of vascular cells [32]. Recently, we identified malondialdehyde (MDA)-adducts as major moieties on circulating MPs, and found that they are largely responsible for their pro-inflammatory activities. Notably, platelet activation via various triggers has been found to result in MDA-generation. Natural IgM antibodies (NAb) as well as complement factor H (CFH) bind MPs via MDA-adducts, promote their clearance, and neutralize their activities [49, 77]. Although low levels of IgM Abs to MDA-LDL are linked to CVD [78-80], it is not known if these IgM actively modulate inflammation and thrombosis. Moreover, a genetic variant of CFH (rs1061170) implicated in age-related macula degeneration impairs its ability to bind MDA-adducts [49]. Since MDA-binding sites in CFH are hot spots of disease associated mutations, other genetic variants affecting CFH binding to MDA likely exist. Hypotheses and Aims: It is hypothesized that MDA-adducts on circulating MPs are critical mediators of inflammation and thrombosis, and that specific IgM NAbs as well as CFH can modulate the activities of MPs. Aim 1. To map the thrombogenic and inflammatory signature of endothelial cells in response to MDA-positive MPs from various cellular sources. Expression patterns of thrombotic/ inflammatory genes induced by MPs and synthetic MDA-adducts will be assessed, and the effect of MDA-specific mAbs and CFH will be tested in vitro. Gene expression and proteomic profiles will be generated, and data integrated for multiplex network analyses. Aim 2. To test the ability of MDA-specific IgM mAbs and CFH to interfere with pro-thrombotic and pro-inflammatory effects of MPs in vivo. Cytokine production, thrombin generation, as well as thrombus formation will be assessed following infusion of MDA-positive MPs into different mouse models, and the neutralizing effect of IgM NAbs and CFH evaluated. In addition, levels of MDA-positive MPs, MDA-specific IgM, and the CFH binding to MDA will be evaluated in plasma of patients with high pro-thrombotic states. Aim 3. To identify mutations in CFH and CFH related proteins that affect MP- and MDA-binding. Patient plasma will be screened for the capacity of CFH to bind MDA by established assays to identify patients with low/high CFH binding capacity. The corresponding genetic variations will be determined using whole genome sequencing. Effects of genetic variants on inflammation and thrombosis will be validated in vitro and in vivo. Methodology: MP assays; FACS; qPCR, ELISA, thrombin generation assay; mouse models; histology and immunohistochemistry; core units for genomics/sequencing, bioinformatics, and cell sorting will be utilized. Synergies with other groups: Expression analyses of MDA- and MP-activated ECs will be done with de Martin, Zellner, Schmid (NFkB signaling) and Thurner; MP characterization of monocyte subsets and platelets with Wojta and Zellner; in vivo assays for thrombosis and inflammation with Lang (splenectomy model) and Knapp (TFfl/fl mice); MP-reactive IgM mAb will be tested with Pabinger and Jilma, novel candidate SNPs will be analyzed with Mannhalter; clinical samples will be provided by Pabinger, Lang and Jilma (endotoxinemia).
Brostjan Christine
Project title: The impact of neutrophil-mediated proteolysis on the pro-thrombotic function of TSP-1
Background: Thrombospondin-1 (TSP-1) is a high-molecular weight molecule primarily secreted by platelets and endothelial cells upon cell activation [1]. It promotes thrombus formation by stabilizing van Willebrand factor (vWF) and platelet aggregates as reflected by enhanced thrombus detachment in TSP-1 k.o. mice [2]. Furthermore, the TSP-1 gene polymorphism N700S is a known risk factor for coronary artery disease and myocardial infarction [3]. The full-length (185 kDa) TSP-1 molecule is susceptible to cleavage by extracellular proteases which generate fragments of distinct function [4-5]. We have found that upon thrombus formation during wound healing, a shift in TSP-1 molecules occurs in human blood from the full-length (185 kDa) to the proteolytically processed (160 kDa) TSP-1 isoform [6]. In vitro co-culture experiments revealed that neutrophil-derived elastase and cathepsin G generate the 160 kDa TSP-1 fragment which constitutes a link between inflammatory activation and thrombosis.
Aims and Methodology: We hypothesize that neutrophil-mediated proteolytic processing of TSP-1 is an essential mechanism promoting the pro-thrombotic activity of TSP-1 and that aberrantly increased proteolysis of TSP-1 contributes to thrombosis. We propose the following experiments to challenge our hypothesis.
We will compare the hemostatic functions of full-length (185 kDa) TSP-1 to the purified 160 kDa TSP-1 isoform by investigating molecule turn-over (cell surface binding, internalization and degradation), the enhancement of platelet aggregation in the presence of TSP-1 variants, and platelet adhesion under flow as a function of vWF cleavage.
We further propose to assess the impact of proteolytic processing on TSP-1 function by comparing a non-cleavable TSP-1 mutant to the wildtype protein with respect to thrombus formation and stability.
In addition, we aim to compare the naturally occurring allelic gene variants N700 and S700 for differences in proteolytic processing and pro-thrombotic function of TSP-1 derived from platelets of genetically distinct donors.
By in vitro co-culture assays we aim to assess whether NET (neutrophil extracellular trap) formation enhances TSP-1 processing by elastase and cathepsin G and results in the local enrichment of proteolytically processed TSP-1 isoforms. We would further like to investigate whether TSP-1 associates with circulating neutrophils and/or NETs detectable in human blood. The NET expertise will be developed together with PI Lang.
In collaboration with PIs Lang and Wojta, we will determine the ratio of 185 to 160 kDa TSP-1 in thrombus material, culprit site blood and peripheral blood of thrombosis patients to evaluate the degree of proteolytic processing in the course of thrombosis. We aim to facilitate sample analysis by the development of a "dual ELISA system" to concomitantly measure the level of 185 and 160 kDa TSP-1.
Based on the SFB-initiated blood sample and data base to identify and characterize high risk thrombosis patients, we will assess whether increased blood levels of 160 kDa TSP-1 are predictive and may identify a high-risk group for arterial or venous thrombosis. TSP-1 isoform levels will be compared with markers of thrombosis as established by PI Pabinger [7], markers of neutrophil activation such as circulating myeloperoxidase and elastase, and with NET frequency in patient blood.
Name of the group leader: de Martin
Project-Title: Activation of endothelial cells by platelets
Background
Beyond their conate role in blood clotting, platelets have been recognized as important contributors to the inflammatory response. They produce several key inflammatory mediators such as platelet-derived growth factors, thrombospondin, P-selectin and interleukins. Activated platelets account for 90% of CD40 ligand in the body due to their ability to shed this molecule, which can directly activate the inflammatory process on the endothelium.
Hypotheses and Aims
Due to expression of CD40 and potentially other membrane-stemming and secreted molecules we hypothesize that activated platelets can evoke an inflammatory response in the endothelium that differs from conventional stimulation by e.g., IL-1, TNFa, or LPS in regard to the gene expression repertoire. This potential specificity of the inflammatory response may in turn provide a handle for specificity of novel therapeutic targets, where one aspect is the identification of negative feedback mechanism. The aims of this project are:
1. to characterize, on the level of gene expression, the response of the endothelium to activated platelets, identify differences to conventional proinflammatory mediators, and identify potential negative regulators. Analyze the involved signaling pathways and transcription factor(s).
2. Test in vitro the identified regulators for their function in endothelial cell activation and coagulation, and select 1-2 most important ones for analysis of their mode of action.
3. Analyze a potential effect of platelets on ECs in two mouse models: a) mice with genetically activated platelets (e.g., constitutive active IKK in megakaryocytes, in collaboration with J. Schmid), b) wild-type mice after intravenous infusion of CD40L.
4. Analyze patient material for the status of the identified regulators in selected diseases.
Methodology
ad 1) Expression profiling (core facility) of HUVEC (available in the lab) with activated platelets and/or shed CD40L (required collaboration). Bioinformatics in part available in lab, but collaboration helpful. Proteomics will be included at a later stage (collaboration required).
ad 2) will be tested by overexpression and knock-down of the respective genes using predominantly inflammatory and coagulation parameters as read-out, technology available, collaboration on specific coagulation assays as read-out required.
ad 3, 4) collaboration required.
Synergies with other groups
1. Available techniques:
Molecular cellular biology techniques including isolation of HUVEC, transfection, viral vectors, analysis of transcription factors and signaling pathways, protein-protein interactions, protein modifications.
2. What is sought from other groups
Expertise on platelets, Clinical samples, mouse models
Cooperations:
Brostjan (or other platelet expert)
Petzelbauer (and other Clinician)
Gerner (or Bioinformatics)
Schmid (mouse models)
Gremmel, Thomas (Co-Investigator: Assinger, Alice)
Project-Title: Influence of new antiplatelet and antithrombotic agents on platelet activation, leukocyte-platelet interaction and platelet-mediated inflammatory processes
Background: Upon activation, degranulated platelets rapidly form heterotypic leukocyte-platelet aggregates (LPA), which represent a very sensitive marker of in vivo platelet activation in several pathophysiological circumstances including myocardial infarction.1-3 Besides procoagulant activity, LPA formation has proinflammatory effects, linking coagulation and development of atherosclerosis4-7, and making reduction of LPA a potential therapeutic goal. However, not all platelet inhibitors are able to diminish LPA formation.8-10
Hypotheses and aims: Newly emerging antiplatelet and antithrombotic drugs yielded promising results in patients´ outcomes in large clinical trials but the underlying mechanisms are not completely understood. We hypothesize that these beneficial effects are partly mediated by a reduction of leukocyte-platelet interaction and platelet-mediated inflammatory processes. Therefore, we aim at studying the effects of new P2Y12 receptor antagonists, thrombin receptor antagonists, Factor Xa inhibitors and direct thrombin inhibitors on platelet activation, leukocyte-platelet interactions and proinflammatory effects of platelets in patients with stable and unstable atherosclerotic vascular disease as well as in experimental animal models.
Methodology: Patient samples will be analyzed by several techniques and technologies: Platelet reactivity testing, platelet adhesion and aggregation assays, FACS analysis, IF, ELISAs, and genotyping of P-selectin and PSGL-1 gene polymorphisms. Moreover, a biobank with plasma and platelet samples will be generated. Pharmacological interventions will be further evaluated in in vitro studies (cell cultures, FACS, IF, qPCR, recruitment assays) and murine atherosclerotic models. Synergies with other groups: We will provide the SFB members our broad experience with FACS analysis especially regarding platelet-leukocyte interaction analysis and leukocyte subset analysis (JILMA /WOJTA), platelet function assessment, leukocyte recruitment and transmigration assays and genotyping of P-selectin and PSGL-1 gene polymorphisms (JILMA). Further, we will generate plasma and platelet samples for the biobank, which will permit thrombospondin isoform testing (BROSTJAN), analysis of microparticle profiles (BINDER), platelet proteomics analysis (ZELLNER) and study of platelet effects on endothelial cells (DE MARTIN).
Jilma, Bernd
Project-Title: Influence of P2Y12 and PAR1 on the platelet leukocyte interaction, microparticle formation & on platelet proteome during endotoxin (LPS) induced coagulation, and staphylococcemia.
Background
The inflammatory and procoagulant host response to infection are intricately linked and platelet-leukocyte interactions appear to play a central role in the pathogenesis of septic shock and ensuing disseminated intravascular coagulation (DIC). It is unclear whether platelets stimulate leukocytes by heteronymous aggregate formation or by the direct release of different mediators. The role of the P2Y12 and PAR1 receptors in the generation of platelet leukocyte aggregates, microparticles, intercellular tissue factor transfer in endotoxin (LPS) induced coagulation are ill-defined.
Hypotheses and Aims
The P2Y12 receptor contributes to platelet-leukocyte interactions in vitro mediated by platelet P-selectin exposure, which induces expression of tissue factor and mediates TF-transfer to the platelets [1, 2]. P2Y12 inhibition diminished the exposure of TF on platelet-leukocyte aggregates, and mitigated intravascular coagulation in mice [3]. We hypothesize that blockade of P2Y12 or PAR1 may blunt TF-triggered coagulation in humans, by reducing platelet activation, platelet-leukocyte interactions, and microparticles, and that endotoxemia and bacteremia may change the platelet proteome. We further hypothesize that PAR1 may influence protein C activation, which could affect the anticoagulant properties of the endothelial cells, and induce endothelial-epithelial barrier disruption, inflammatory cell recruitment, and fibrin deposition.
Methodology
Human models of endotoxemia [4] and lung inflammation have been established by the applicant. The trials will be performed according to inter/national guidelines and applicable regulations. Further confirmation shall be obtained from animal models of Gram positive septicemia, and clinical trials in patients with bacteremia. Outcome parameters will be quantified by previously established assays.
Synergies with other groups
Unique samples plasma, platelet samples and bronchoalveolar lavage from endotoxemic and/or bacteremic animals, healthy volunteers and patients will be generated and will be accessible to all members of the consortium to validate biomarkers, e.g. malondialdehyde carrying microparticles (Binder). Samples will permit testing of thrombospondin isoform function and release can be tested under stress conditions of increased platelet secretion, augmented elastase and enhanced VWF release by Brostjan. Changes in the platelet proteome and secretome will be analyzed in collaboration with methods established by Zellner et al..
Knapp, Silvia (Co-Investigator: Gernot Schabbauer)
Collaborator: Nigel Mackman, University of North Carolina at Chapel Hill, N.C., U.S.
Project-Title: Cell-type specific impact of tissue factor on the crosstalk between lung-inflammation and thrombosis
Background: Inflammation and coagulation are closely linked as both pathways influence and activate each other. Bacterial products, cytokines or complement proteins induce expression of tissue factor (TF), which is the initiator of the coagulation cascade resulting in thrombin activation. Thrombin in turn contributes to inflammation via protease activating receptors (PAR). We and others could demonstrate that monocyte-expressed TF amplifies inflammation and worsens outcome in murine sepsis and endotoxemia models [95]. Beside the importance of the inflammation-coagulation crosstalk during systemic infections, local inflammation of lungs has been shown to be accompanied by activation of coagulation, although the underlying mechanisms and consequences are less well understood.
Hypotheses and Aims: Studies on TF-mediated effects were so far limited to the use of inhibitors because TF-KO mice are not viable [96]. We will now make use of only recently generated conditional TF-knock-out mice crossed with cell-type-specific Cre-recombinase mice that will help us understand the contribution of the respective cellular TF source to pulmonary inflammation and coagulation [95, 97]. To understand the precise role of TF-mediated pathways within the lungs, we will study models of sterile lung injury - where we expect advantageous effects in the absence of TF - and models of clinically relevant bacterial pneumonia, such as induced by Streptococcus pneumoniae - where TF-mediated inflammation might prove beneficial by augmenting inflammation and possibly by trapping and killing of bacteria in clots. The specific aims are:
1) To evaluate the effect of macrophage- or epithelia-specific TF-deletion on the regulation of coagulation, as well as on the inflammatory and thrombotic response during sterile acute lung injury and pneumococcal pneumonia. 2) To investigate the impact of microparticles and oxidized lipids on TF-mediated inflammation and coagulation during lung injury. 3) To investigate selected bacterial mutants with respect to the generation of fibrin traps in lungs.
Methodology: Mouse strains with cell type specific deletions via the Cre-recombinase system are available as well as murine models of acute lung injury and bacterial pneumonia. Techniques to analyze inflammation and anti-bacterial effector mechanisms are established.
Synergies with other groups: We offer TF fl/fl mice as a tool to ablate initiation of coagulation in a cell type specific manner (to P. Petzelbauer) and can provide animal models of sterile inflammation and infections to study coagulation-associated functions of leukocytes and/or platelets (for C. Mannhalter). Microparticle isolation and analysis will be done by C. Binder and C. Mannhalter. Analysis of lipid peroxidation products in alveolar lavage fluids is planned to be performed in collaboration with the group of Valery Bochkov (external)
Lang, Irene
Project-Title: Innate immune cells and thrombus resolution
Background: Thrombosis involves shedding of microparticles and debris from inflammatory cells. Recent studies suggest that these products of cellular turnover play an integral part in the inflammatory component of thrombosis and may amplify thrombus formation (1). Engulfment of apoptotic cells (efferocytosis), membrane blebs (macropinocytosis), protein ubiquitinylation, protein sumoylation, and cleavage of nuclear or cytoplasmic proteins are main mechanism restoring tissue homeostasis (2). We have established a mouse model of stagnant flow venous thrombosis, and utilized splenectomy to ablate cells of the innate immune system as potential mediators of efferocytosis. As expected, thrombi of splenectomized mice were significantly larger than those of controls (ANOVA, n=8, p<0.03).
Hypotheses and Aims: Our data demonstrate that innate immune cells play a key role in thrombus resolution. We hypothesize that these cells mediate efferocytosis, which is required for thrombus resolution and restoration of vascular patency. Therefore, the aims of this project are:
to identify the cell subset that mediates thrombus resolution we will selectively reconstitute B-cells, T-cells and monocyte/macrophages
to delineate how these cells mediate efferocytosis
to analyse thrombus resolution in mice in which Mertk, a tyrosine kinase receptor for the phosphatidylserine-binding protein Gas6, which bridges apoptotic cells to phagocytes is defective (3);
to compare gene/protein expression arrays of thrombi after splenectomy versus thrombi after reinfusion of splenic cell subsets to understand specific mechanisms of efferocytosis
Methodology: Several techniques and technologies will be employed: ELISA, FACS analysis of thrombus, Gene/ Protein expression profiling (Core facility Genomics), immunohistochemistry, proteomics, Western blot, PCR, angiogenesis assays (Matrigel, BrDu, sprouting), murine KO models (e.g., conditional flk-1-/-, CD31-/-, Mertk-/-)
1) Contribution to the SFB-Vascular Biorepository: We are able to provide plasmas and cells of patients after splenectomy, and of controls (ethics approval number 307/2003). We hold several mouse strains including flk-/- and CD31-/- mice and have experience in experimental venous thrombosis. We have broad experience with FACS, molecular biology techniques, immunohistochemistry and ELISA. We are able to perform primary cell cultures from patient samples.
2) Expected from the outside: Functional assays (Brostjan, Wojta), proteomic techniques, data processing and systems biology.
Pabinger, Ingrid (Co-investigator: Cihan Ay)
Title: Coagulatory / inflammatory phenotypes in diseases with risk of venous and arterial thromboembolism
Background: Patients with cancer and those with antiphospholipid syndrome have a high risk for venous and arterial thromboembolism, and have also been found to exhibit an increased inflammatory status. In the Vienna Cancer and Thrombosis Study (CATS), which is an ongoing prospective study, we could recently demonstrate that both activation of coagulation and parameters of increased inflammation are strong and reproducible risk factors for future venous thromboembolism in cancer patients [101-104]. Another group of patients with an increased risk for venous and arterial thromboembolism are patients with antiphospholipid antibodies. Patients with an acute venous thrombotic event and without another disease also show activation of coagulation and increased inflammation, which gradually decrease over time. Circulating microparticles [105] have been reported to bear tissue factor and other procoagulant proteins and thus should be studied systematically in patients with cancer and antiphospholipid antibodies. Furthermore, it has been shown that structural and functional fibrin clot abnormalities [106] are associated with arterial and venous thrombosis and with inflammatory diseases, but were to date not studied prospectively in cancer and antiphospholipid syndrome patients. Hypotheses and Aims: To study the relationship of tissue factor bearing microparticles and structural and functional fibrin clot properties with markers of inflammation (CRP, blood count parameters, fibrinogen-levels) and platelet activation (sP-selectin) and to investigate their predictive role for future venous and arterial thrombosis in cancer and the antiphospholipid syndrome. In addition samples from our cohort will be studied by other members of the SFB. Methodology: Clinical studies: Ongoing recruitment into the prospective observational cohort studies in two groups of patients with increased inflammatory potential and a high risk of venous thromboembolism and longitudinal study of patients with and acute venous thromboembolic event. We will analyse 1) Patients with newly diagnosed cancer (1600 patients, ongoing); 2) Patients with antiphospholipid antibodies (150 patients, ongoing); 3) Patients with acute venous thrombosis will be recruited at presentation and followed with repeated blood sampling over a period of 6 months; 4) A healthy control group of at least 100 individuals to assess normal values of parameters. Due to the ongoing patient-recruitment and repeated sampling, specific material, as needed by other members of the SFB, can be obtained during blood collection. Laboratory analysis will include 1) Tissue factor bearing microparticles (already established in our lab); 2) Structural and functional fibrin clot properties (pore size of the fibrin clot, fibrin clot compaction, turbidity measurement, enzymatic fibrin clot stability). Synergies with other groups: Samples will be used for the laboratory tests listed and in other laboratories assigned to this SFB (e.g. C.Mannhalter, J.Schmid, H. Wojta, C. Brostjan). Findings can then be linked to the extent of inflammation and the clinical manifestations of venous or arterial thromboembolism, in addition the predictive value in the whole patient-group and subgroups of patients can be evaluated. Parameters available in our patient groups, such as D-dimer, prothrombin fragment 1+2, sP-selectin, factor VIII, CRP and IL-6 will also be determined in the newly recruited patients and used in multivariable models. We will offer determination of tissue factor bearing microparticels and fibrin clot properties to other members of the SFB.
Petzelbauer, Peter
Project-Title: Thrombomodulin as a target to prevent re-stenosis in vascular grafts
Background: The success of percutaneous coronary intervention and bypass surgery is often limited by neointimal hyperplasia causing arteriosclerotic obliterating re-stenosis. Postoperative down regulation of endothelial thrombomodulin (TM) expression is linked with the induction of neointimal hyperplasia. TM contributes to vascular thromboresistance through complex formation with thrombin followed by protein C activation. Genetic polymorphism of TM correlates with the risk of thrombotic diseases and recombinant TM has been shown to improve the long-term patency of stent grafts in animal models. Pharmacological inhibition of TM down regulation can be achieved by inhibition of RhoGTPases, Retinoic acid, ß-adrenoceptor antagonists and statins, but their relevance for prevention of TM down regulation and re-stenosis is only partly resolved [107-111].
Hypotheses: Inhibition of RhoGTPase activation maintains TM surface expression and counteracts intima hyperplasia and re-stenosis in venous-arterial grafts.
Aims: 1) to test the effect of dominant negative Rho constructs and of various RhoGTPase antagonists on endothelial TM expression in vitro (under basal conditions and in situations of TM suppression as e.g., induced by TNF). Questions include, which RhoGTPase is involved, which GEF activates the respective RhoGTPase, to which scaffold protein is this GEF associated. 2) To test the effect of RhoGTPase antagonists on TM expression, protein C activation, inflammation and intima-hyperplasia in syngeneic and allogeneic saphena-carotis interponats in mice. Questions include: a) Can adenoviral over expression of TM in vascular grafts improve outcomes, whereas TM inhibition by over expression of TMshRNA results in the opposite effect? b) What is the effect of tissue factor knock down in combination with Rho-inhibition? c) Can TM expression be maintained by therapeutic RhoGTPase inhibition?
Methodology: Cell culture of endothelium and SMC, inhibitors of small G proteins of the Rho family (peptide Bbeta15-42; US and EU patent, inventor PP), cingulin-derived peptides (European patent application, inventor PP 2010), Rac1 Inhibitor W56, statins, etc.), dominant negative Rho constructs, fusion proteins (biosensors) to test site-specific Rho activation by FRET technique in living cells; animal model for vena cava grafts into the carotid artery as in-vivo model for neointimal hyperplasia.
Synergies with other groups: An animal model for vena cava grafts into the carotid artery as in-vivo model for neointimal hyperplasia to analyze inflammatory infiltrates is established (Wojta); the FRET technique to quantify Rho activation in living cells will be further analyzed with Thurner/Hanel, inflamed tissue samples can be studied in collaboration with de Martin and endothelial co-culture systems with Lang. We plan to collaborate with Knapp for defining the role of TF in protective effects of TM by using TF -/-mice; with Brostjan for analysis of TSP-1 fragments in venous grafts (TSP-induced chemotaxis on smooth muscle depends on GTPase activity, TSP-1 fragments in re-stenosis not defined), with Jilma to analyze blood coagulation parameters in mouse and with Mannhalter to measure P-selectin serum levels and platelet activation.
Schmid, Johannes A.
Project-Title: The role of the inflammatory molecule IKK2 in atherothrombosis
Background: Several proteins regulating coagulation such as tissue factor, adhesion molecules, as well as components of the complement system are controlled or influenced by the NF-kappa B signaling pathway [51, 112, 113]. These intracellular signaling pathways are furthermore pivotal for the intercellular crosstalk between endothelial cells, leukocytes, platelets and smooth muscle cells in the course of inflammation-associated thrombosis. Most of the signals leading to NF-kappa B activation converge at the I-kappa B kinase complex, with IκB kinase 2 (IKK2) as a central component in the activation pathway [114].
Hypotheses and Aims: We hypothesize that IKK2 has a central role in thrombotic events triggered by persistent inflammation or stress conditions. Based on the fact that aortic thrombosis is a frequent clinical problem we aim at establishing two transgene mouse models providing a) an inducible, aortic endothelial cell specific expression of constitutive active IKK2 and b) a megakaryocyte-specific expression of this enzyme. These mouse models should be exploited to investigate the effect of IKK2 (and constitutive NF-kappa B activation) on the expression of pro-thrombotic genes and the feedback circuits within the inflammatory signaling pathway network both on the level of endothelial cells and on the level of megakaryocytes and platelets. We intend to study that alone or on an ApoE-/- background at a cholesterol-high diet. Furthermore we plan to complement the mouse models with cell culture studies of human aortic endothelial cells and with high content image analyses of patient material.
Methodology: A mouse strain with a constitutive active IKK2 silenced by a loxP-flanked Stop cassette is available [115]. Furthermore, we have access to a tamoxifen-inducible, aortic endothelial cell-specific Cre mouse line (Bmx-CreERT2, unpublished, provided by Christian Weber), allowing for a timed control of EC- specific Cre. A mouse line expressing megakaryocyte-specific Cre recombinase [116] will be obtained from the group of R. Parilla. Analyses of these mouse models will be complemented with cell culture experiments using primary cells transfected with lentiviral expression of suppression constructs and with novel high content imaging microscopy of patient material using automated cell recognition and single cell-resolution tissue cytometry.
Synergies with other groups: Cooperations will be done predominantly with the groups of R. de Martin (on NF-kappa B signaling after activation of endothelial cells with platelets), with the group of Gerner (to analyze the proteome of platelets, megakaryocytes and endothelial cells from our transgene mice), with the group of Thurner for network analyses and systems biology evaluation of the observed changes; with the group of Knapp for cross-breeding of transgene mouse strains and analyses of mouse phenotypes, with the groups of Lang and Pabinger to obtain patient samples for atherothrombosis; with the group of Mannhalter to study platelet function and the role of NF-kappa B in regulating protein synthesis or mRNA degradation in platelets; and with the group of Wojta to study the impact of the NF-kappa B pathway for the establishment of different coagulatory macrophage phenotypes and in order to obtain primary cells for lentiviral transduction experiments.
Wojta, Johann (Co-Investigator: Walter Speidl)
Project-Title: Coagulatory phenotypes of monocyte and macrophage subsets
Background: Inflammation and coagulation play key roles in atherosclerosis and in particular in the development of its late complication namely atherothrombosis.[117, 118] Monocytes and macrophages participate critically as effector- as well as as target-cells in these processes and are centrally involved in linking inflammation and coagulation.[119] Recently, subsets of monocytes and macrophages, each with characteristic pro- and antiinflammatory properties, have been described.[120] Circulating Ly-6Chi monocytes in mice and CD14+CD16+ monocytes in humans are proinflammatory and patients with coronary artery disease have higher numbers of CD14+CD16+ monocytes than healthy controls.[121] In contrast, Ly-6Clo and CD14+CD16- monocytes may have anti-inflammatory properties and are decreased upon hyperlipidemia.[122, 123] It had also been shown that atherosclerotic plaques contain macrophages of the proinflammatory M1 and the antiinflammatory M2 phenotype.[124] However, divergent coagulatory phenotypes of these macrophage and monocyte subsets have not been characterized yet.
Hypotheses and Aims: We hypothesize that - in analogy to the modulation of inflammatory events - different subsets of monocytes and macrophages differently participate in the regulation of coagulation. We will pursue the following aims: 1. We will characterize such coagulatory phenotypes of different monocyte subsets as effector cells in thrombus formation and thrombus dissolution in humans and mice in vivo and in vitro by studying the constitutive and regulated expression of molecules involved in these processes (e.g. TF, uPA, PAI-1, MMPs) in monocyte subsets obtained from healthy controls, patients with stable coronary artery disease and patients with acute myocardial infarction and from control and ApoE-/- mice. 2. We will investigate monocyte subsets as a source of thrombogenic microparticles. 3. We will study subsets of monocytes and macrophages as target cells in coagulation by analyzing the constitutive expression of PARs in these cells and by examining the effect of procoagulant stimulation by aggregate-formation of monocytes with platelets and by stimulation of monocytes and macrophages with thrombin on the expression of molecules involved in the modulation of inflammation and thrombus formation and dissolution in these cells. 4. We will characterize coagulatory phenotypes of macrophages and foam cells originating from different monocyte subsets in vitro as outlined under aim 1 and 2 for monocytes. 5. We will aim to detect monocyte and macrophage subsets in atherosclerotic plaque specimens and coronary thrombi and analyze the expression of molecules involved in thrombus formation and thrombus dissolution.
Methodology and synergies: We have available in our group: Cell culture techniques; isolation and characterization of monocyte subsets; macrophage and foam cell formation; immunohistochemistry; RT-PCR; ELISA. We expect from outside: FACS of monocyte subsets (Core Facility Flow Cytometry); advanced immunohistochemistry (Core Facility Imaging); gene arrays (Core Facility Genomics); proteomics and bioinformatics (platform)
