Thrombosis Is A Hallmark Of Vascular Disease Biology Essay

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Recent evidence suggests that inflammation and aberrant immune reactivity, which are associated with the recruitment of specific cell types, are strong drivers of thrombosis. However, the organ and disease-specific mechanisms are poorly understood for venous thromboembolism, arterial thrombosis as it occurs in acute coronary syndrome, peripheral artery disease, stroke, or on vascular grafts. A strong association of these conditions is evident from recent trials [1] and epidemiological studies [2]. Patients with acute coronary syndromes or stroke have an increased susceptibility to developing venous thromboembolism as a complication of hospitalization [3] and patients with inflammatory conditions are at risk to suffer cardiovascular events [1, 4]. Endothelial injury as a trigger for atherothrombosis is well established, as has been demonstrated by electron microscopy of deep veins [5]. ???better examples ?

Atherosclerosis is an inflammatory disease that involves the arterial wall and is characterized by the progressive accumulation of lipids in the vessel wall. The first step is the deposition of lipids (LDL) in the intima and endothelial cell activation, which enhances the permeability of the endothelial layer and the expression of cytokines/chemokines and adhesion molecules. Subsequently, LDL particle accumulate in the extracellular matrix where they become targets for oxidative and enzymatic modifications. In turn, retained pro-atherogenic LDL enhances selective leukocyte recruitment and attachment to the endothelial layer inducing their transmigration across the endothelium into the intima. Monocytes differentiate into macrophages, a process associated with the upregulation of Toll-like receptors leading to foam cell formation. Foam cells release growth factors, cytokines, metalloproteinases and reactive oxygen species all of which perpetuate and amplify the vascular remodelling process. In addition, macrophages release tissue factor (TF) which, upon plaque rupture, contributes to thrombus formation. Platelets recognize ligands in the ruptured or eroded atherosclerotic plaque, become activated and aggregate, leading to thrombosis and to the clinical manifestation of the atherothrombotic disease. Acute thrombus formation on disrupted atherosclerotic plaques plays a key role during the onset of acute coronary syndromes. It is currently believed that lesion disruption facilitates the interaction between circulating blood and TF within the atherosclerotic lesion. Vessel-wall TF has been traditionally considered the major determinant of thrombosis. However, this old dogma has been recently challenged owing to the discovery of a pool of TF that circulates in blood. Several studies have shown that circulating TF is associated with monocytes, granulocytes and platelets in cell-derived microparticles, and occurs as a soluble protein generated by alternative splicing of its full-length mRNA. Recently, increased circulating TF activity has been associated with a hypercoagulable state, leading to the concept of 'vulnerable blood'. Part of the blood-borne TF circulates in an 'inactive' form and needs to be 'activated' to exert its thrombogenic potential. Recent evidence suggests that increased circulating TF activity may potentiate the initial thrombogenic stimulus represented by vessel wall-associated TF, leading to the formation of larger and/or more stable thrombi. Inflammation plays a key role by increasing TF expression and activity. In turn, TF upregulation may facilitate inflammation by enhancing intravascular fibrin deposition, formation of proinflammatory fragments of fibrin, and by generating coagulation proteases, including FVIIa, FXa and thrombin. A functional link between the blood clotting cascade and the inflammatory response was apparently favored in evolution of higher organisms given that tissue injury requires an arrest of bleeding as well as the defense of invading microorganisms. However, the basic co-operation of these two stress-response systems can be detrimental as for instance in patients with endotoxemic shock, where the blood coagulation system is hyperactivated. Thus, the relationship between coagulation and innate immunity is of profound impact and therapeutic interest.

Cellular Mediators involved in inflammation and thrombosis

Despite being anucleated cells, platelets belong to the major players in thrombosis, inflammation and related diseases. During blood coagulation, platelets release pro-coagulatory molecules like van Willebrand factor and thrombospondin-1 (TSP-1) and rapidly form cell aggregates. They are also an important source of pro-inflammatory molecules e.g. P-selectin, TF, CD40L, cytokines, chemokines or metalloproteinases. According to recent findings, platelets are also important for the innate immune response and combat infections. During systemic inflammation, upon stimulation with pathogens or their toxins, platelets secrete a vast variety and amount of pro-inflammatory and procoagulant factors, and by membrane budding they give rise to microparticles (MPs). In response to LPS, stimulated human platelets secrete microparticles that contain mature IL-1β newly synthesized in platelets [11].

Platelets not only express complex patterns of surface proteins regulating cell-cell interactions, they also secrete a rich repertoire of functional agonists including pro-inflammatory compounds. Currently, little is known how the expression of these important players is regulated in vivo. While certain platelet responses such as degranulation can occur within seconds, other functional adaptions may take hours, and it is fair to expect that subtle biological adjustments may take even days. While all kinds of short-term responses of platelets have been well investigated, the subtle capabilities of platelet proteome changes to enter pro- or anti-inflammatory cell states are not fully understood [12].  Adequacy of platelet inhibition is a contemporary issue in patients, as inter-individual variability in drug response has been reported.

A growing number of patients with atherosclerotic vascular disease receive chronic therapy with novel ADP receptor antagonists and new anticoagulants, which led to promising results in patient survival in large clinical trials. However, the effects of these new agents on platelet cytokine release and leukocyte-platelet interactions are unclear.

In addition to platelets there is another set of anucleated membrane compartments, which has a central role in thrombosis, designated as microparticles (MPs). These are small and heterogeneous membrane vesicles defined by a size of 0.1 - 1.0 µm, which usually expose a negatively charged phosphatidyl-serine-rich surface [13-15]. They are released from platelets, endothelial cells, leukocytes, erythrocytes, or tumor cells in response to apoptosis or cell activation [16, 17]. MPs are present in the plasma of healthy individuals [16-18], and elevated levels have been found in inflammatory, metabolic, malignant and thrombotic diseases [17], acute coronary syndrome and acute pulmonary embolism [19-23]. In an experimental mouse model of venous thrombosis platelet-derived MPs were found to promote thrombus formation [24]. Recent studies have demonstrated increased levels of circulating MPs, especially of tissue factor (TF)-bearing MPs in venous thrombosis patients, patients suffering from rheumatoid arthritis, systemic lupus erythomatosus, and primary Sjögren's syndrome [25]. However, data from prospective clinical studies are scarce and the clinical significance of MPs as a useful biomarker for prediction of venous and arterial thromboembolism still remains to be elucidated. Furthermore, a functional classification into different groups or subsets of origin has not been done so far, which complicates the interpretation of published studies.

The pro-coagulant property of MPs is largely mediated by the presence of TF, which is the main initiator of the blood coagulation system in vivo. However, the presence of phosphatidyl-serine as well as other phospholipid-derived components on the surface of MPs has also been shown to enhance coagulation [26-28]. MPs can activate cells through specific interactions with cell surface receptors due to a high concentration of ligand molecules which they carry on their surface, or through direct transfer of cell-derived components to recipient cells thereby modulating their function [29-32]. For example, MPs isolated from human atherosclerotic plaques promote monocyte adhesion on the endothelium by direct transfer of ICAM-1 to endothelial cells [33]. Platelet-derived MPs have been shown to promote inflammation in arthritis by triggering cytokine responses from synovial fibroblasts in an IL-1 dependent manner [34]. In a recent studyit was demonstrated that monocytic MPs activate endothelial cells by transferring IL-1β along with other components of the inflammasome [35]. Finally, oxidized phospholipids and other lipid peroxidation derived structures in membranes of MPs have also been shown to promote inflammatory responses in endothelial cells [36, 37]. This is of particular interest as these moieties are likely to be shared among MPs of different cellular origin. Physiological clearance mechanisms that target MPs and mediate their removal from the circulation exist to maintain homeostasis [38]. In addition, natural IgM antibodies and other soluble pattern recognition receptors have been implicated in the clearance of MPs [39, 40] as well as in MP-associated complement activation [41]. Indeed, apoptotic cells and MPs are recognized by natural IgM antibodiesand complement factor H (CFH), via the recognition of specific lipid peroxidation derived structures present on their surfaces [42-45]. These structures represent danger-associated molecular patterns that are recognized by various arms of innate immunity [46]. However, the role of these specific immune responses in MP-mediated inflammation and thrombosis is unknown and impaired clearance mechanisms may be key modulators of MP functions.

In conclusion, modulating the pro-inflammatory and pro-thrombotic activities of MPs as well as modulating MP clearance may provide novel points of therapeutic interventions in chronic inflammatory diseases and thrombosis.

Besides platelets and microparticles endothelial cells (ECs) have a central role both in inflammatory processes and in thrombosis. A great variety of stimuli can activate ECs and trigger an inflammatory response predominantly via the transcription factor NF-κB shifting haemostasis in most cases towards a pro-coagulant state. These stimuli include cytokines such as TNFα or IL-1β, bacterial cell wall components of the LPS family, signaling molecules like CD40L, mechanical stress, or viruses and pathogens activating the Toll-like receptors [47]. Most of these signals converge at the level of the I-kappa B kinase complex (IKK), which phosphorylates the inhibitory molecules of the I-kappa B family, leading to their poly-ubiquitination and proteasomal degradation thereby releasing NF-κB as active transcription factor. Active NF-κB then induces the expression of adhesion molecules on the cell surface, which trigger binding of platelets and leukocytes, as well as the expression of anti-apoptotic genes and cytokines. Finally, NF-κB also drives the expression of anti-inflammatory genes, which control stress response under physiological conditions. However, many diseases or pathological conditions are characterized by insufficient negative feedback mechanisms leading to chronic inflammatory states. The clinical consequences may be severe given that the inflammatory state of ECs and other cell types involved, is also shifting the equilibrium of haemostasis towards a pro-coagulant state. NF-κB can induce procoagulant molecules including tissue factor and adhesion molecules, while anti-coagulant molecules are often downregulated [48]. Recently it became evident that pro-coagulatory molecules exhibit pro-inflammatory properties [48], which might lead to a vicious cycle of inflammatory and thrombotic reactions, as exemplified in its extreme by the disseminated intravascular coagulation (DIC) in the course of sepsis.

This interrelation is also reflected by the fact that anti-inflammatory drugs may reduce thrombotic events - and that anti-coagulant agents such as activated protein C (APC) attenuate inflammation by blocking nuclear translocation of NF-κB [49]. An important component in this regard is the anti-coagulatory molecule thrombomodulin, which is expressed on ECs depending on shear forces and which is downregulated by inflammatory mediators [50]. Currently it is not understood in detail how these signaling networks are tightly controlled and which processes ultimately lead to the complete deregulation and instability of the system towards a chronic or acute pathological state. Many of the feedback circuits, signaling networks and crosstalks are poorly characterized or the acquired knowledge is restricted to qualitative rather than quantitative information about these processes. The latter would be essential for enabling predictions about the specific course of inflammation or thrombosis.

When endothelial damage promotes a prothrombotic state induced by proinflammatory cytokines and adhesion molecules [51], ECs bind not only platelets and microparticles, but also leukocytes, which represent additional important mediators linking inflammation and thrombosis. Atherosclerotic plaques prone to disruption and subsequently thrombosis contain blood-borne immune cells (mainly macrophages and T-lymphocytes).

Macrophages are the predominant immune cells in atherosclerotic lesions. They produce pro-inflammatory cytokines, participate in lipid retention and vascular cell remodeling, and express pattern-recognition receptors. They have the ability to engulf modified LDL and form foam cells [52]. Production of matrix metalloproteinases (MMPs) by macrophages leads to degradation of extracellular matrix thereby weakening the consistency of the fibrous plaque cap. Plaque rupture is then followed by exposure of the potent pro-coagulant protein tissue factor (TF). Macrophages are therefore considered to promote atherosclerosis and atherothrombosis as shown in studies of mice deficient in functional macrophage-colony stimulating factor [53]. In a rabbit model of atherosclerosis it was demonstrated that neutrophil granulocytes contribute to vascular inflammation, however, with less effect than macrophages [54]. While it is known that macrophages can develop into subtypes with distinct pro- and anti-inflammatory properties, the coagulatory phenotypes of macrophages and foam cells originating from different monocyte subsets have not been characterized to date. It seems of particular interest to address the important question how these cells could modulate both thrombosis and thrombus resolution.

B-lymphocytes may inhibit atherothrombosis by production of antibodies against oxLDL and by preventing neointimal hyperplasia and endothelial damage [55]. One project within this consortium will focus on how natural antibodies (NAb) produced by these cells are able to bind microparticles via malondialdehyde adducts, thus actively modulating their thrombogenicity. In a mouse model of experimental atherosclerosis it has been shown that splenectomy promotes disease progression, whereas adoptive transfer of splenic B-lymphocytes seems to have protective effects [56]. Moreover, bone marrow derived B-lymphocytes preserved cardiac function after myocardial infarction by cardiomyocyte salvage in a rat model [57]. In addition, leukocyte recruitment and angiogenesis play a crucial role during thrombus resolution. While neutrophils modulate an inflammatory response by secreting cytokines, proteases, and reactive oxygen species, monocytes are recruited into maturing thrombi over time and thrombus resolution is severely impaired when their recruitment is reduced [58]. Up to now data concerning the role of B-cells in venous thrombus resolution is insufficient, although B-cell participation seems likely. The project of Lang's group intends to crosslink the information derived from atherothrombosis with a venous thrombosis model.

1.2. Specific Aims within the First 4-Year Period

Sub-Group I: pro-inflammatory activities of platelets and MPs

Involved groups: Binder, de Martin, Gremmel, Jilma, Knapp, Pabinger, Schmid, Wojta

Aim 1: Define subsets of MPs

(Binder, Pabinger, Jilma, Wojta,)

MPs are vesicular particles originating from various cellular sources, which are currently only defined according to their size - not taking a potential diversity and heterogeneity sufficiently into account. Our consortium aims for characterizing MPs in more detail using preparations from different sources and analyses by flow cytometry, as well as biochemical assays (Western Blots, ELISAs, 2D-DIGE and MS-analyses). We plan to analyze MPs from the blood circulation of patients (obtained from the groups of Pabinger, Jilma and Lang), from the blood of various mouse strains (groups Binder, Schmid and Knapp), as well as MPs derived from certain cell types, such as macrophages at various differentiation states (group Wojta), neutrophils (group Brostjan), ECs (Binder, de Martin) or isolated platelets (Gremmel and others). Cytometry will be applied to phenotype MPs with respect to the occurrence of important markers (TF, phosphatidylserine on the surface, MDA-adducts, CD40L, P2Y12R etc.) and will be done by the group of Wojta for the different MP sources in parallel in order to achieve comparability. In addition we aim for a more detailed analysis using 2D-DIGE of various MP preparations (Zellner, group Jilma) and mass spectrometry analyses of differential spots (with the help of the proteomics core facility). We expect that this will allow us to define functional subsets of MPs, which might have completely different effects on thrombosis as well as inflammation.

Aim 2: Identify pro-inflammatory and pro-thrombotic effects of MPs

(Binder, Jilma, Knapp, Pabinger, Wojta)

It is the goal of this SFB to identify mechanisms by which MPs mediate their pro-inflammatory and pro-thrombotic properties, and to define the clinical importance in thrombosis. In the first period of this SFB application a detailed characterization of the link between inflammatory and thrombogenic properties of circulating MPs will be provided - ranging from molecular to clinical characterizations. The group of Wojta will assess whether MPs derived from different monocyte subsets differ in their functional activities. The group of S. Knapp will characterize the role of TF+ MPs in models of lung injury. The tight association of MPs with inflammatory markers will be further studied in detail by the group of I. Pabinger, who will evaluate this relationship in large prospective clinical studies focusing on VTE in patients with cancer. The group of C. Binder will focus on the functional role of a subset of MPs that are enriched in pro-inflammatory lipid-peroxidation derived structures, and delineate their role in various clinical cohorts available within this SFB. They will explore the capacity of these MPs to induce a specific response in endothelial cells, and test the ability of natural IgM antibodies as well as CFH to interfere with MP activities. In another approach with potential therapeutic implications, B. Jilma will focus on the capacity of P2Y12 receptor inhibition to prevent the generation of inflammatory and thrombogenic MPs. The detailed characterization of the pro-inflammatory/pro-thrombotic activities of various types of MPs or platelets on endothelial cells will further involve the groups of de Martin (gene expression) and Schmid (NF-κB signaling). Strategies that modulate circulating MP activity might be novel tools of therapeutic intervention to prevent thrombosis in patients at high risk.

Aim 3: Study pro-inflammatory effects of platelets

(de Martin, Gremmel, Jilma, Pabinger, Schmid)

One important goal of our consortium is to elucidate various pro-inflammatory effects of platelets on endothelial cells and leukocytes. One approach will be to investigate endothelial cell activation by platelets at different stages (activated or not) in particular based on their expression of CD40L - and to identify the specific set of target genes as wells as feedback mechanisms triggered by EC-platelet interactions (de Martin).

The group of Schmid will test the impact of constitutive NF-κB activation in megakaryocytes on function and activation of platelets in a transgene mouse model.

Gremmel, Jilma: Pabinger

Aim 4: Investigate potential anti-inflammatory effects of novel anti- platelet drugs

We hypothesize that the beneficial effects of novel antiplatelet drugs and anticoagulants are partly mediated by reduction of platelet-mediated inflammatory processes. Therefore we aim at studying the effects of new P2Y12 and thrombin receptor antagonists, as well as inhibitors of factor Xa and thrombin on leukocyte-platelet interactions and platelet-mediated pro-inflammatory effects in patients with stable and unstable atherosclerotic vascular disease (Gremmel/Assinger). This will be done by determining leukocyte-platelet interactions, cytokine profile and platelet activation profiles. In parallel we plan to determine the underlying mechanisms of the inhibitory effects on platelet-mediated inflammation in in vitro inhibitor studies. The obtained results should be further confirmed in experimental animal models

Sub-Group II: pro-thrombotic activities of leukocytes and ECs

Involved groups: Brostjan, Knapp, Lang, Petzelbauer, Schmid, Wojta, de Martin, Gremmel

Aim 5: Investigation of pro-thrombotic effects of leukocytes and ECs

The group focusing on leukocytes and thrombosis will address selected topics in the areas of atherothrombosis and venous thrombosis, referring to individual cell types that have been implicated in specific functions. It is intended to investigate 1) coagulatory phenotypes of monocyte and macrophage subsets both in animal models and in human subjects (Wojta); 2) the impact of neutrophil-mediated proteolysis on the prothrombotic function of thrombospondin-1 (Brostjan), 3) the subsets of B-lymphocytes that mediate thrombus resolution in an animal model of slow-flow venous thrombosis, and in human subjects (Lang); and 4) the impact of macrophage-specific deletion of TF on the crosstalk between lung inflammation and thrombosis (Knapp).,

Furthermore, we plan to clarify the role of IKK2 and the expected persistent activation of the NF-κB pathway on thrombosis using transgene mouse models, where we express constitutively active IKK2 in an inducible manner in aortic endothelial cells (Schmid), Additionally, we intend to study the therapeutic potential of Rho-GTPase inhibition to counteract restenosis based on the observation that restenosis after vascular grafting is linked to downregulation of thrombomodulin, which appears to be controlled by Rho-GTPase (Petzelbauer).

Aim 6: Establishment and initial usage of a vascular biology specific bio-databank

1.3. Definition of Long-Term Goals


1.3.1.Platelets and Microparticles



Furthermore, the roles of inhibiting P2Y12 and protease receptors in systemic coagulation will be characterized, which should clarify whether they represent potential therapeutic targets in disseminated intravascular coagulation (Jilma ?).


Our longterm goal is to determine the superiority of pharmacological targets in different clinical settings to guarantee an optimal outcome in patient treatment. A better understanding of the underlying mechanisms and effects of platelet inhibitors as well as a better understanding of interindividual variability in drug response can provide important new knowledge for improved therapeutic strategies and individualized antithrombotic therapy (Gremmel).

1.3.3. Leukocytes

The consortium will expand their observations to human subjects. Wojta and colleagues will analyze peripheral blood from patients with acute coronary syndromes, and expand their analyses to atherosclerotic plaque tissue harvested during carotid endarterectomies. Lang's group will exploit an existing clinical and plasma/cellular/DNA database of patients after splenectomy (ethics approval number 307/2003) and search for B-lymphocytes and B-lymphocyte subsets in freshly harvested samples from these patients. Brostjan et al will compare the naturally occurring allelic gene variants N700 and S700 for differences in proteolytic processing and function of TSP-1 derived from platelets of genetically distinct donors. In a next step, clinical follow-up data from defined patient subsets will be collected and related to proteomic/genetic patterns of cellular activation.

1.3.2. Endothelial Cells

The work on the mutual crosstalk between ECs and other cell types involved in inflammation and thrombosis will be continued. On the long-term, we strive for a better understanding, how inflammatory and thrombotic processes are interconnected and which feedback circuits might be crucial in a clinical context. We hypothesize that currently used drugs have important and maybe beneficial off-target effects, e.g. with anti-inflammatory compounds having effects on thrombosis and vice versa. Moreover, we believe that drug combination strategies targeting both the inflammatory and the thrombosis branch of a disease might exhibit synergistic effects. Therefore, we aim for a better elucidation of the signaling networks and feedback circuits not only on a qualitative but also on a quantitative level. We want to achieve this goal for the intracellular signaling networks of the different cell types involved, but also for the intercellular communication and signaling circuits. In this context, it will be extremely important, to combine the expertise of the involved groups for the different cell types and to complement it with state-of-the-art bioinformatics and furthermore with complex systems biology approaches. By including analyses of a sufficient number of patients, we hope to provide a basis for personalized medicine in inflammatory diseases, combining quantitative multi-parameter analyses with predictions on effects of drug combination therapies.

1. 4. Coherence of the Different Projects and Synergies

An added value of the research will be achieved by linking the different projects synergistically and by providing a vivid information flux between the groups involved. State-of-the-art bioinformatics and systems biology will be exploited to compare data of the different projects and to generate a more complete picture of the scientific field.

Our central goals to reach a better understanding of the complex interactions between cells and their signaling networks in inflammation and thrombosis cannot be achieved with single, separate projects but only with a tight network of research initiatives linked by an intensive communication and interaction of the scientists involved. Furthermore, our combination of clinical and basic research groups will provide an excellent platform to combine patient-relevant aspects with the study of molecular mechanisms of disease processes. Our consortium provides an excellent combination of experts for different cell types, various diseases, distinct methodologies and complementary perspectives in the fields of inflammation and thrombosis. Synergies will be achieved not only by the combination of complementary techniques and know-how but also by a systematic sharing of resources such as primary or immortalized cells, transgenic mouse models, patient samples and data, as well as proteomics or micro-array data. Furthermore, the groups will be able to utilize the core facilities for proteomics, flow cytometry, imaging and genomics of the MedUni-Vienna. Details of the synergies and collaborations are specified in the abstracts of the single groups. A graphical illustration of the interactions and synergies between the groups is shown in the Appendix section on page 132).


Collaboration of PhD's and Postdocs

Local collaborations

Thurner: Complex Systems group

Gerner (Med. Univ. Wien): For bioinformatics of proteomics …

International collaborations

Are briefly mentioned in the CVs of the group leaders - and omitted here for reasons of space restrictions

Nigel Mackman (Schabbauer, Knapp, Lang)..

Bernhagen (Schmid)

1.5. Expected Advancement in Science and Importance for the Scientific Community

We expect that the combination of our expertise in the field of thrombosis and chronic as well as acute inflammation research sets the stage for major advancement in the understanding of the cellular interactions and feedback circuits linking inflammation and thrombosis. We are confident that the synergistic mixture of competence for different cell types and the link between clinical and basic research, as well as the combination with complex systems biology approaches will lead to a substantial added value and the prospect to make a significant contribution to the international scientific community. The results are expected to pave the way for new therapy approaches and more systematic strategies to combat inflammatory and thrombosis diseases.

The ultimate goal of the consortium is to understand cellular triggers and modulators of vascular thrombosis, and eventually, vascular occlusion. This is a relevant problem affecting both the arterial and the venous systems. Advances in the understanding of the role of cellular components will 1) help identify new treatment targets; 2) disclose modes of drug delivery and 3) permit the targeted application of treatments that modulate vascular patency.

The expected scientific outcome of this project proposal - contributing to a better understanding of the influence of inflammation on thrombosis in the venous or arterial circulatory systems - is of highest relevance as thrombotic events are major causes of death in the western world.

Open Source aspect of the bio-databank: microarray, proteomics data, image datasets, anonymized patient data etc: will be provided in an accessible databank on the website of the consortium.

1.6. Gender-Relevant Aspects of the Research

It is known that both inflammation and thrombosis [59-61] exhibit significant gender-specific differences. An increased risk for VTE as well as arterial thrombosis has for example been described in women on estrogen and progesterone therapy [62]. On the other hand, atherothrombotic diseases are primarily found in male patients at young age, while the risk for women to develop cardiovascular diseases increases to a comparable level after menopause. Our joint effort to elucidate mechanisms of and crosstalk between inflammation and thrombosis promises to unravel gender-specific differences in this respect, as the consortium has access to a high number of patients and a highly professional proteomics, bioinformatics and systems biology.

2. Human Resources

2.1. Expertise of the Group Leaders and Infrastructure

(alphabetically ordered according to the last names)

Christoph Binder is an expert on the role of the immune system in atherosclerosis. C. Binder first described the atheroprotective role of the natural IgM antibody T15/EO6 [63], which was followed by studies defining its protective mechanisms [36, 64], and identified the atheroprotective role of IL-5 in mice and then humans [65, 66]. Recently, his group demonstrated that oxidation-specific malondialdehyde epitopes are major targets of natural IgM antibodies in mice and humans [43], which was followed by the discovery that complement factor H specifically binds malondialdehyde epitopes and protects from oxidative stress [45]. Experimental approaches involve immunological techniques, various mouse models of atherosclerosis, and the analyses of patient samples. Major techniques used include adoptive in vivo cell transfer and bone marrow transplantation; lipoprotein and microparticle isolation and the biochemical generation of oxidized lipids. The team of Christine Brostjan has extensive experience in endothelial cell research, including the isolation and culture of primary human endothelial cells from different organs, and state-of-the-art molecular and cellular biology techniques to investigate endothelial cell activation, function and gene regulation (including gene transfer and silencing technologies). Furthermore, a particular interest in the interactions of endothelial cells with other blood cell populations has led to the establishment of in vitro co-culture assays of endothelial cells, platelets, PBMCs, and granulocytes which will be available to SFB members for the characterization of inflammatory reactions related to thrombosis. With respect to clinical investigations, C. Brostjan has optimized and applied techniques for blood sampling without in vitro platelet activation, which will prove valuable in assessing parameters of thrombotic events in patients. The group of Rainer de Martin has extensive expertise in molecular and cellular biology of the endothelium with a focus on the molecular regulation of inflammation and, in particular, the mechanisms of activation and de-activation of NF-κB. In this context his group is interested in negative feedback regulation in inflammation. Using transcriptomic analyses after different inflammatory stimuli he could identify novel feedback mechanisms of NF-κB signaling as well as crosstalk to other signaling processes such as the MAPK or JNK-pathways. Furthermore, his group is interested in biological consequences of NF-κB activation such as anti-apoptotic and cell survival mechanisms. The infrastructure of the group includes all necessary equipment for molecular biology and cell biology of endothelial cells.

Thomas Gremmel is a specialist in internal medicine at the Division of Angiology of the Department of Internal Medicine II at the MedUni-Vienna. He has broad experience in antiplatelet and anticoagulant therapy in cardiovascular disease and venous thromboembolism. In recent years, he established various platelet-function tests for the assessment of residual platelet reactivity. In his research, he focuses on platelet response to antiplatelet therapy, leukocyte-platelet interactions and genetic polymorphisms influencing coagulation and platelet activation. All equipment that is essential for the proposed experiments is available. This equipment includes a VerifyNow® platelet function analyzer, an APACT 4S plus aggregometer, a flow cytometer and a high sensitivity Western Blot imaging device. Alice Assinger, the co-investigator of T. Gremmel, has an expertise in platelet function analysis and platelet-leukocyte interactions in different inflammatory models. She recently returned to the Center for Physiology and Pharmacology of the MedUni-Vienna, from a 1-year postdoctoral position with Cecilia Söderberg-Naucler at the Centre for Molecular Medicine at the Karolinska Institute in Sweden. During her time at the Karolinska Institute A. Assinger worked on the role of viral infections in atherosclerosis and widened her experience with animal studies, platelet-leukocyte interactions as well as in vitro leukocyte recruitment-flow models.

Bernd Jilma has extensive expertise in conducting clinical pharmacology trials including a large human endotoxemia study. Another major part of his work is dedicated to studying the regulation of coagulation, von Willebrand factor, and platelet function in various patient groups. More recently, new animal models of bacteremia and of disseminated intravascular coagulation have been established, and staphylothrombin has been cloned and expressed. The Department of Clinical Pharmacology is composed of a clinical research ward and several laboratories. Full platelet biology equipment is available, including video capillary microscopy, calibrated automated thrombogram, rotational thrombelastometry analyzers and platelet function analyzers including multiple electrode impedance aggregometry. Maria Zellner as co-investigator of B. Jilma is very experienced in sample preparations for proteome analysis with a special focus on standardized protein extraction methods for clinical studies followed by 2D-electrophoresis and mass spectrometry analysis. She discovered and validated several clinical biomarkers and achieved their translation into routine analysis methods such as ELISAs or protein chips. As a result of these activities she holds patents for biomarkers and currently develops a platelet-protein biochip. The group of Zellner has access to high-standard proteomics technologies including fluorescence 2D gel electrophoresis and scanning, SDS-PAGE/LC-MS/MS of tryptic digested peptides and instrumentation for functional platelet analysis.

Sylvia Knapp is an expert in the innate immune response to clinically relevant bacterial infections such as pneumonia as well as in sterile inflammations including acute lung injury and additionally works on the crosstalk between lung-inflammation and thrombosis. She is interested in the molecules involved in the initiation and resolution of the innate immune response to pathogens and on the role of bacterial virulence factors and their interactions with host structures and pathways. She demonstrated that oxidized phospholipids negatively impact host defense against E. coli in vivo. The expertise of her laboratory includes: in vitro and in vivo bacterial infection and inflammation models; basic cellular and molecular biology techniques, biochemical analysis of signal transduction pathways, RNAi approaches, cytometry, in vitro immunological assays and basic confocal microscopy. Supporting infrastructure available in her laboratory comprises: qPCR, fluorometer, Licor Odyssey imaging system, Amaxa nucleofector and facilities for immunohistochemistry, experimental animal work, as well as biosafety level-2 workbenches (including space allocated for infectious diseases). Her co-investigator, Gernot Schabbauer, started out doing research on the regulation of tissue factor and the coagulation cascade by mitogenic as well as inflammatory stimuli. He has expertise in the research of murine innate immunity models and macrophage biology. Furthermore, he is an expert in the analysis of the role of the PI3-Kinase signaling pathway in the regulation of coagulation and inflammation.

Irene Lang leads a vascular biology research group in Austria since 1994, when she returned from a 5-year postdoctoral fellowship at the Committee on Vascular Biology of the Scripps Research Institute, USA. Since 2004 she holds a professorship for Vascular Biology at the MedUni-Vienna. The focus of her research has been vascular occlusion as a sequela of thrombosis. While chronic thromboembolic pulmonary hypertension serves as a clinical model for her research, the group employs a mouse model of stagnant flow venous thrombosis for the simulation of thrombus formation and resolution. One of her approaches is the sequential knock-out of molecules involved in angiogenesis and inflammatory cell recruitment and their impact on thrombus resolution, which is considered a stimulus for the initiation of a "vascular occlusive" genetic program. I. Lang is designated as vice-speaker of the consortium. She is also serving as executive for the ongoing establishment of a cluster for cardiovascular medicine (CCVM) at the MedUni-Vienna and she is deputy director of the university's PhD programme.

Ingrid Pabinger is very experienced at designing, organizing and performing clinical studies on patient groups with an increased risk for venous and arterial thromboembolism. Furthermore, I. Pabinger has extensive experience in the field of functional hemostatic tests and ELISA techniques. The group has ELISA readers and devices for performing hemostatic tests, a fully automated coagulation analyzer for clotting, chromogenic and turbidimetric assays (Ceveron alpha) and a fluorometer. Freezers for collecting samples are available and in addition part of the samples is stored in the Biobank run by the central laboratory of MedUni-Vienna and the general hospital Vienna.

Peter Petzelbauer is an expert in endothelial cell biology; he discovered a novel function of a vasculo-protective peptide with anti-RhoA activity [67, 68] filed a patent and succeeded in performing a phase II study in humans showing the efficacy of this peptide in patients with myocardial infarction undergoing percutaneous catheter interventions [69]. The group has experience with FRET technology for GTPase-biosensors to measure Rho activity.

Johannes Schmid has extensive experience in intracellular signal transduction with an emphasis on inflammatory processes and the NF-κB activation pathway. J. Schmid holds two different university lecture qualifications (habilitations); one for the field of vascular biology (at the MedUni-Vienna) and one for biochemistry (at the University of Applied Life Sciences and Natural Resources, Vienna). He is experienced in a variety of molecular, cellular and biochemical techniques and uses state-of-the-art microscopy and quantitative image analysis for studying the dynamics and interactions of signaling molecules. Furthermore, he has substantial experience with transgene mouse models. In reference to his function as consortium speaker, he started accumulating a management experience in 2004, when he was co-founder of the new Ludwig Boltzmann Institute of Cancer Research, for which he served as deputy director and chief financial officer in addition to heading a group focusing on cooperativity between inflammation and cancer. He further developed his organizational skills by professional seminars on: project management, leading scientific research teams, optimizing performance, conflict management, team development as well as management of institutes. The group of J. Schmid is located at the pre-clinical campus and has access to the entire infrastructure necessary for the project.

The group of Johann Wojta offers expertise in the isolation, characterization and cultivation of various human primary cells involved in cardiovascular pathologies, such as endothelial and smooth muscle cells from various vascular beds, monocytes and macrophages or cardiac myocytes and fibroblasts. The group also has expertise in employing these cells in various bio-assays including adhesion assays under static and flow conditions, as well as transmigration assays. The laboratory also has experience in protein immune- and histochemistry, protein analysis, ELISA and real-time PCR.

2.2. Gender and Child Care Issues

Our SFB-consortium considers itself as a network of scientists, where expertise and competence are appreciated irrespective of gender. The proportion of women as group leaders in the consortium is 36.4%, all of them having an outstanding scientific reputation and productivity. Young scientists will be recruited and supported on an equal opportunity basis. We aim at reaching a percentage of 50% female researchers for Postdocs and PhD students. Facilities for child care are available at the MedUni-Vienna.

2.3. Support of Young Scientists

An important aim of the consortium is to support and promote young gifted scientists by making use of the interdisciplinary and interactive research program. The distinct but overlapping expertise of the different groups will be exploited for educational purposes to allow for a multi-disciplinary training. The recruitment of PhD students and Postdocs will be done by a coordinated effort following an international announcement; invitation of candidates for hearings and a professional selection process. We intend to establish a laboratory-rotation system for PhD-students working in similar thematic topics. We expect that this will lead to direct personal links between the groups, thereby improving communication as well as transfer of expertise. Since our university offers a well-established international PhD program for the field of Vascular Biology we will encourage all our PhD students to enroll in that particular program. Furthermore, we will propose that our students and postdocs establish a thematic group within the Young Scientist Association (YSA, of the MedUni-Vienna. The YSA organizes a PhD symposium with international keynote lecturers on a yearly basis, which has developed to a well-established forum of scientific communication and exchange. Five members of our consortium (Binder, Brostjan, Lang, Petzelbauer and Wojta) are also involved in the application for a funded doctoral program headed by Dr. Johann Wojta (title: "Translational Research - Bridging Basic Research and Patients' Needs"). In case that both applications are granted we will interact strongly and synergistically with this program, which would help to build up a significant critical mass.

Link to the PhD program:

3. Impact on the Austrian Science System

3.1. Visibility of the Planned Research

A vivid participation in the national and international scientific community is already a self-evident element of the current groups and will be further driven towards a better visibility of the consortium. In this context we aim at developing a "corporate identity" by using a common layout and name in presentations or talks. We intend to publish the results of our research activities with an open-access policy - by making use of the collaboration between the Austrian Science Fund, FWF, and the UK PubMed Central archive. In this case a potential open access fee is covered by the FWF for most of the journals. Besides participating in the scientific community via publications or conferences, we intend to establish also a more general, popular dissemination strategy to become visible for the general audience. This will be achieved on several levels, including a website explaining aspects of inflammation and thrombosis in a popular scientific way and providing links for patients to appropriate medical institutions. Second, we intend to write articles for popular scientific magazines, which are accessible to patients in the waiting rooms of physicians or ambulances. These activities will be coordinated by a secretary to be employed by the consortium. All group leaders will be asked to contribute appropriate scientific text and media for these purposes.

Tag der offenen Tür

(annual open house event)

3.2. Commitment of the Research Organization (MedUni-Vienna)

The MedUni-Vienna has a strong interest in the establishment of the proposed research network and will support the consortium in several ways: 1) by granting access to the core facilities of the university charging only costs for consumables but not personnel or infrastructure costs; by providing the necessary infrastructure, laboratory and office resources and by offering administrative support, as for instance regarding IT-requirements for setting up a website and an intranet platform. In addition, the university offers a broad spectrum of courses for personnel development. The strategic development plan of the MedUni-Vienna includes a focus on vascular biology, and an emphasis on immunology - therefore, the present application is entirely in line with the profile building process of the university. A majority of the groups are located in the newly opened Anna Spiegel Research Building of the MedUni-Vienna ( which is equipped with state-of-the-art cell culture facilities, an SPF-mouse facility, an isotope laboratory, a histology unit, a FACS-Calibur flow cytometer, chemiluminescent and fluorescent readers, a Biacore instrument, ultracentrifuges, as well as excellent infrastructure for molecular biology, biochemistry, immunology and cell biology techniques. A high-end flow activated cell sorting facility, an advanced imaging facility, and a next generation sequencing unit as well as mass spectrometry equipment are available as core facilities and are accessible to consortium members.

Raumkonzept, CCVM, DK, Profilbildung im Gebiet Vaskuläre Medizin gemäß des Entwicklungsplans

4. Organization and Financial Plan

4.1. Financial Plan (see Appendix)

Our financial concept is based on a personnel budget covering two scientists (2 PhD students or 1 postdoctoral fellow and a part-time technician) per group and a consumable budget of about 15.000 € per scientist and year. In addition, we consider a budget for mouse facilities and general project costs covering travel expenses and costs for dissemination activities.

4.2. Internal Processes and Structures for Communication, Cooperation and Decision Making

The executive board comprises all group leaders, and will meet on a monthly basis with the speaker (or the vice-speaker) serving as moderators and organizers. These meetings will provide an information platform to monitor the general progress and to discuss organizational and administrative aspects (as for instance laboratory rotation of students; in detail planning of collaborations etc.). Financial issues within the group budgets as well as personnel decisions will fall within the responsibility of the single group leaders as long as they do not affect the other groups. In case that superordinate financial or administrative decisions have to be made, they will be decided by a simple majority of the executive board. The speaker and the vice-speaker will meet on a weekly basis to discuss organizational questions that affect the whole consortium. The speaker will work on a daily basis with the secretary to handle administrative tasks such as setting up and running an intranet platform, financial controlling or establishing a reagent and ordering platform. The secretary will help in disseminating the research results and in maintaining contact to press and media. For internal communication we will setup a password-protected intranet platform on a secure server, where we will deposit information on resources such as DNA constructs, antibodies, cell lines, mouse strains, patient cohorts (anonymized) as well as data from experiments or studies and publications. We plan to produce a monthly newsletter, which will describe achievements, social events, and internal affairs and which is intended to contribute to the team spirit of the consortium. On a yearly basis we will organize a research retreat to present and discuss the scientific results of the groups. In addition, we will also organize a social event once per year to support team building and personal links between the groups.

Research Retreats


Datenbank von Reagenzien (AK, Mausstämme,), Protokolldatenbank


Biodatenbank (Blut etc, auch Infos).

Ideen: Fotos der Leute….

Website mit Links der Mitglieder

Notes to the Reviewers

The applicants would like to state that the proposed joint project has a significant chance for being selected for the 2nd application phase only in case of a very high ranking (above 93%). So even if the proposal gets a good review it might not have a chance for a detailed elaboration in a second phase if the score is below 93%. This statement is not intended to put any pressure on the reviewers and we are certainly willing to accept any constructive feedback. We just wanted to state that if the reviewers think that this project proposal should make it to the main selection phase, it will be necessary to rate it accordingly.