Key words:
Leukocyte-Endothelial-Interactions, Inflammation, Cell Adhesion, Selectins,
Fucosyltransferases, Glycosylation, Cytokines, Cadherins, Catenins
Research in our department is focused on various aspects of vascular cell biology and concentrates mainly on the molecular basis of the interactions of leukocytes with the vascular sytem, which control leukocyte migration and extravasation as the basis for inflammatory reactions and lymphocyte surveillance of the organism. In addition, we work on novel molecular mechanisms potentially involved in the formation of the cardiovascular system.
Leukocyte extravasation is usually triggered by pathological stimuli such as physical injury or infectious microorganisms and is the starting event that initiates the process of inflammation and that controls the continuation of inflammatory reactions. Understanding its molecular basis will allow to interfere with and modulate the often harmful consequences of the inflammatory process. Immune surveillance is the second process that is based on the extravasation of leukocytes. Lymphocytes continuously enter lymphatic tissue (lymphocyte homing) while they are patroling the organism. Similar mechanisms that allow lymphoid and myeloid cells to migrate through the body are probably also used by metastatic cells.
Leukocyte extravasation is controlled by molecular factors that activate the endothelium and the leukocytes and by adhesion molecules that mediate the contact between both cell types. Selectins, a small family of three adhesion molecules (L-, E- and P-selectin) that bind carbohydrate ligands usually initiate the contact between leukocytes and endothelial cells, leading to leukocyte-rolling on the blood vessel wall. This enables the leukocytes to sense chemotactic factors such as chemokines on the surface of the endothelium, which leads to the activation of leukocyte integrins. Binding of the integrins to their endothelial ligands, mainly members of the immunoglobulin supergene family, allows leukocytes to firmly adhere to the blood vessel wall and to actively migrate towards a source of chemotactic factors. Following this selectin-, chemokine-, integrin-driven cascade of molecular interactions, leukocytes finally transmigrate through the blood vessel wall by a largely unknown mechanism. Since many years or group has been analyzing the ligands of the endothelial selectins (PSGL-1 and ESL-1) (Lenter et al. J. Cell Biol. 1994, 125, 471; Steegmaier et al., Nature 1995, 373, 615; Borges et al., 1997, 90, 1934; Hidalgo et al., Immunity, 2007, 26: 477).
In addition we clarified the molecular basis of the human genetic disease called leukocyte adhesion deficiency II (LAD-II). We found that it is based on a mutation in the GDP-fucose transporter that leads to a lack of fucosylation and thereby, among other defects to a lack of selectin ligands, the basis for the immunodeficiency (Luhn et al., Nature Genetics 2001, 28: 69). Together with clinicians at our University in Münster we could establish the first successful therapy (a fucose substitution therapy) that cured the immunodeficiency defect in LADII patients (Marquardt et al., Blood 1999, 94:3976; Luhn et al., Blood 2001, 97:330).
Since 1990 our group has focused on molecular mechanisms that control leukocyte capturing, concentrating on the selectins and their ligands. This topic is still actively investigated. Since a few years a major part of our activities is now directed towards tackling the question, how leukocytes actually move through and overcome the blood vessel barrier. Even basic questions such as whether leukocytes would move directly through an endothelial cell, i.e. by transcytosis or whether leukocytes squeeze through inter-endothelial cell junctions is still not settled and actively debated. We favor the idea that leukocytes move through endothelial junctions and we follow the working hypothesis that leukocytes become activated by binding to adhesion molecules and chemokines present on the endothelial cell surface, while endothelial cells are probably triggered by yet unknown signals from the leukocytes that lead to the opening of interendothelial junctions. In this context we are interested in the molecular mechanisms that control the opening and re-formation of endothelial cell contacts during the process of leukocyte extravasation (diapedesis).
Therefore, we have started to systematically analyse molecules that control diapedesis and that mediate or control inter-endothelial cell contacts. It is likely that some of our studies on these mechanisms will also generate knowledge that will be relevant beyond the process of leukocyte extravasation and extend into the area of angiogenesis, blood vessel formation and remodelling. We are mainly concentrating on three classes of molecules in this context:
1) JAMs and related proteins: Second, we study a group of tight junction associated membrane proteins that belong to a subclass of two Ig-domain proteins. A prototype of these adhesion molecules was originally designated as junctional adhesion molecule (JAM) and that is now called JAM-A. This is a member of the Immunoglobulin-supergene family (Ig-SF) with two Ig domains of the V and C2 type. Two close relatives, JAM-B and JAM-C are also found at endothelial tight junctions, but the three JAMs are not specific for endothelium. Beside the JAMs we are analysing the structurally more distantly related Ig-SF members endothelial cell-selective adhesion molecule (ESAM). We identified ESAM by a classical mAb approach searching for novel endothelial cell contact proteins (Nasdala et al., J. Biol. Chem., 2002, 277: 16294). Recently we showed that ESAM is required for neutrophil extravasation. In addition, VEGF induced endothelial permeability is reduced in ESAM-deficient mice and ESAM supports Rho activation. Our results suggest that ESAM is involved in leukocyte as well as VEGF-induced opening of endothelial cell contacts (Wegmann et al., J. Exp. Med. 2006, 203: 1671). Coxsackievirus Adenovirus Receptor (CAR), is the closest relative to ESAM. Its found on neuronal cells, various epithelia and on embryonic cardiomyocytes. CAR-deficient mice are embryonic lethal (E13.5) due to defects in cardiac development (Dorner et al., J. Cell Sci. 2005, 118:3509). Orientation and bundling of myofibrils in cardiomyocytes was diorganized.
2) VE-cadherin and associated proteins: VE-cadherin is the endothelial specific cadherin that is essential for endothelial cell contact stability. We have identified a receptor type tyrosine phosphatase (VE-PTP) Baumer et al., Blood 2006, 107:4754) as binding partner of VE-cadherin that enhances the adhesive activity of VE-cadherin in transfected cells (Nawroth et al., EMBO J. 2002, 21: 4885). VE-PTP is the first known endothelial specific tyrosine phosphatase. Gene disruption for VE-PTP leads to embryonic lethality at E9.5 due to defects in vascular remodeling (Baumer et al., Blood 2006, 107:4754). Besides VE-PTP, we found a second novel binding partner of VE-cadherin, the cytosolic kinase Csk (C-terminal Src kinase) (Baumeister et al., EMBO J. 2005, 24:1686). We found that Csk binds to phosphorylated tyrosine residue 685 in the cytoplasmic tail of VE-cadherin. This binding enables Csk to inhibit VE-cadherin associated Src and reduce endothelial cell proliferation.
3) CD99 and CD99L2: We have recently found that CD99, a protein that belongs to no known family of membrane proteins, is involved in lymphocyte and neutrophil migration into inflamed tissue in vivo (Bixel et al., Blood 2004, 104:3205). In addition we have now found a distantly related protein, CD99L2 that participates in neutrophil but not in lymphocyte extravasation (Bixel et al., Blood 2007, 109: 5327). Both proteins are found at endothelial cell contacts as well as on leukocytes, however it seems to be the endothelial forms of each of the two proteins that are involved in the extravasation process. Furthermore, we could demonstrate by intravital and electron microscopy that it is the diapedesis process that is affected by antibodies against CD99 and CD99L2.
A major goal of our studies will be to reveal the mechanisms by which endothelial cell layers are triggered to support leukocyte extravasation. Mechanisms believed to be essential in this process would need to be tested in transgenic mice that are mutated on the basis of our understanding of the molecular principles. Our present working hypothesis predicts that leukocytes trigger active intracellular processes in endothelial cells that lead to a controlled opening of the endothelial cell contacts. In addition, contacts between endothelial cells and leukocytes most likely activate proteolytic or other mechanisms that help to overcome the basal membrane. The candidate molecules believed to be involved in the process of diapedesis are VE-cadherin and associated factors that influence its activity, Ig-SF proteins such as JAMs and ESAM and other membrane proteins such as CD99 and CD99L2.
In addition to these major goals, we will sytematically study the molecular mechanisms by which endothelial cell contacts influence angiogenesis. The interactions between VE-cadherin and the vascular endothelial growth factor receptor-2 (VEGF-R2) on the one hand and the vital role of the VE-cadherin associated endothelial specific receptor-protein tyrosine phosphatase VE-PTP during embryonic development (Baumer et al., Blood 2006, 107:4754) on the other hand are basic targets in this context. The interaction of the cytosolic kinase Csk with VE-cadherin and its role in the control of endothelial cell proliferation (Baumeister et al., EMBO J. 2005, 24:1686) is another way by which VE-cadherin may affect the formation of blood vessels.