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Workshop: Intravital Microscopy and Live Cell Imaging in Angiogenesis Research Friday, July 18, 2003, 09.00
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L1 Application of optical and transfection techniques from cell physiology to intact resistance arteries Steffen-Sebastian Bolz & Ulrich Pohl Institute of Physiology, Ludwig Maximilians University, Munich, Germany (E-mail: [email protected]) The linkage of vascular genes to specific functions is a prerequisite for a better understanding of cardiovascular pathophysiology. Cultured vascular cells are an unsatisfying model to analyse gene function because they are separated from their natural environment and their multilateral interaction with other cell types. Moreover, the uptake of plasmid DNA using non-viral techniques is usually low in these cultured cells. The aim was, therefore, to study cell signalling and the effects of an altered gene expression in vascular cells in intact arteries. We established a non-viral experimental model that allows for highly efficient transfection of VSMCs in isolated functionally intact resistance arteries that, in contrast to cultured cells, also allow direct assessment of vascular functions. The core of the model are resistance arteries isolated from the hamster gracilis muscle which are cannulated and continuously perfused with an optimized culture medium for up to 3 days without loss of endothelial or smooth muscle function. During the culture protocol vessels were exposed to a mixture of GFP (green fluorescent protein)-coding plasmid DNA and the non-liposomal lipid transfection reagent Effectene
to visualize transfection efficacy. To indicate functionally relevant gene expression of proteins in VSMCs, active, inactive or GFP-tagged mutants of different signalling proteins of the RhoA pathway that plays an important role for the regulation of microvascular tone were transfected. Subsequently, changes in VSMC Ca2+ (Fura 2method), vascular diameter (videotechnique) and distribution of GFP-tagged RhoA or the enzyme myosin light chain phosphatase (MLCP, immunostaining) in response to the RhoA-dependent vasoconstrictor sphingosine-1phosphate (S1P) have been studied. The monitoring of S1P-induced translocations of the cytosolic proteins RhoA and MLCP to the plasmamembrane by immunostaining substituted for classical biochemical methods to determine protein enzyme activity that were not applicable because of the small sample size. The expression of the alien protein GFP that was homogeneous throughout the VSMC layers of the vessel wall did not affect endothelial or smooth muscle functions, thereby allowing for a proper control for functional effects after expression of proteins of the Rho signalling pathway. Transfection of a dominant negative RhoA mutant or bacterial C3 transferase that specifically blocks RhoA completely inhibited S1P-induced constrictions. Evidence from optical and functional tests thus indicated that isolated resistance arteries have been successfully transfected with a much higher efficiency than isolated vascular cells. The presented techniques allow gene functions to be assessed in direct conjunction with signalling pathways in intact vascular tissue and provide a new tool for microvascular proteomics. The highly efficient transfection will allow to introduce optical protein-based techniques like FRET into this model to directly monitor activity changes of enzymes involved in the regulation of microvascular tone.

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L2 Intravital videomicroscopy techniques in angiogenesis research Ian C. MacDonald, Alan C. Groom & Ann F. Chambers Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada (E-mail: [email protected]) Background: The endothelium is a single layer of cells that lines the blood and lymphatic vessels. Because the endothelial cells of capillaries comprise little more than two lipid bilayers, with thickened regions for the nucleus, they are difficult to resolve in histological sections. Intravital microscopy, however, reveals such vessels by the movement of red blood cells through them and by differences in refractive index between the wall and adjacent structures. Even in thick tissues such as spleen where absorption of light cannot be used to form an image, refraction of light at cell membrane interfaces can be used to produce high resolution images of subcellular features. Materials and methods: Intact organs of anesthetized small rodents are exposed and positioned on a coverglass window on the stage of an inverted microscope. Oblique transillumination from a fiber optic source is deflected as it passes through unstained structures with varying refractive indices. Light intensity is increased on one side of the structure and decreased on the other, giving a shadowed effect which can be used in combination with fluorescence imaging. High numerical aperture lenses are used to optically section the tissue so only those features within a narrow depth of focus are resolved. Light from out of focus layers adds evenly to the background of video images and is removed by adjusting brightness and contrast. Results: Endothelial walls of small blood and lymphatic vessels (up to 50 lm from the surface) can be resolved and the movement of cells within them can be seen and recorded for analysis. Endothelial interactions with circulating cells have been resolved in organs such as spleen, liver, lung and muscle, and in tumors where angiogenesis and poorly formed vessels are seen. Conclusions: Intravital microscopy can be used to study endothelial development and vascular interactions within intact organs and tissues of living preparations.

L3 Intravital fluorescence videomicroscopy to study tumor angiogenesis and microcirculation Peter Vajkoczy Department of Neurosurgery, Klinikum Mannheim, University of Heidelberg, Germany (E-mail: [email protected]) Mechanistic analysis of tumor angiogenesis requires sophisticated in vivo experimental models and techniques. Intravital fluoresecence videomicroscopy is currently the only technique that allows a direct assessment of tumor angiogenesis, microcirculation, angioarchitecture and overall perfusion. Advanced optical equipment has significantly improved imaging quality at up to 1000-fold magnification using both trans- and epi-illumination techniques. The strength of the technique is that it provides a direct, continuous, and non-invasive approach to directly visualize the tumor microvasculature. With this, the technique is superior to other techniques that are currently applied to in vivo microciculation research. Also, the intravital fluorescence videomicroscopic technique enables for quantitative analysis of tumor microhemodynamic parameters. This appears mandatory since nutritive tissue perfusion is not only reflected by morphological parameters, but involves also microhemodynamics and rheology. Finally, intravital fluorescence videomicroscopy provides information on molecular transport and delivery, intra- and extravascular cell-to-cell and cell-to-matrix interaction, as well as tumor oxygenation and metabolism. The aim of the this presentation is to offer a brief overview on the development and present status of intravital fluorescence videomicroscopy. Different examples on how this technique has been

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applied will demonstrate why intravital fluorescence videomicroscopy represents a powerful experimental tool to study tumor angiogenesis and microcirculation.

L4 Intravital microscopy to study leukocyte-vessel wall interactions in tumor microvasculature Arjan W. Griffioen Angiogenesis Laboratory, Research Institute for Growth and Development (GROW), Department of Pathology, Maastricht University and University Hospital, P.O. Box 5800, 6229 Hx Maastricht, The Netherlands (E-mail: [email protected]) The expression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions is suppressed in malignancies. In the present study, we investigated in vivo the regulation of leukocyte-vessel wall interactions by the presence of a tumor. For this purpose we developed a non-invasive intravital microscopy technique for longitudinal studies during tumor development. This technique will be presented in detail. Using this technique TNFa-stimulated leukocyte-vessel wall interactions were studied in ear skin microvessels of nude mice bearing small human LS174T colon carcinomas and in C57Bl/6 mice bearing murine B16F10 melanomas. Leukocyte-vessel wall interactions were studied both within and outside small tumors growing in the ear, and in ear microvessels of mice with a large tumor growing on their flank. Tumor-free mice were used as controls. Compared to values measured at the edge of the ear and in the contralateral ear, leukocyte adhesion was found to be significantly diminished in vessels inside the ear tumor in both mouse models. This reduction disappeared with increasing distance from the tumor. Surprisingly, the level of leukocyte adhesion in ear venules of mice with a large flank tumor was also significantly reduced. Leukocyte rolling, i.e. the step preceding adhesion, was not influenced by the presence of a tumor in nude mice, but was downregulated in immune competent C57Bl/6 mice. Treatment of mice bearing a small ear tumor with a humanized anti-VEGF antibody prevented the dowregulation of leukocyte-vessel wall interactions inside the tumor vessels compared to the nontreated group. FACS-analysis showed that isolated tumor ECs have suppressed levels of ICAM-1 as compared to ECs from normal mouse tissues. In cultured b.END5 cells the TNFa-induced upregulation of ICAM-1 and VCAM-1 was reduced in ECs that were pre-incubated with bFGF or VEGF. The current results may have an impact on the effectiveness of clinical immunotherapeutic treatment protocols.

L5 Studying vesicular coat assembly and membrane interactions in living cells with sub-resolution microscopy techniques Rainer Pepperkok Advanced Light Microscopy Facility and Cell Biology and Cell Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany (E-mail: [email protected]) Background: The vesicular coat complex COPII is involved in transport vesicle formation at the endoplasmic reticulum. It consists of a small GTPase Sar1 and two further sub-complexes sec23/24 and sec13/31. Several homologues of the sub-complexes exist in mammalian cells, which raises the possibility of different COPII complexes existing in parallel in the same cell. Consistent with this we could recently identify with multicolor timelapse microscopy a COPII, COPI and microtubule dependent sorting step at the ER exit site level suggesting that distinct COPII complexes might operate on distinct transport cargo molecules. Materials and methods: In order to test this question directly and to understand the underlying molecular mechanisms for such cargo sorting we have GFP-tagged several components of the COPII coat and

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investigated their kinetics of membrane interactions and participation in COPII assembly by using fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) in living cells. Results: Our data show that COPII components cycle on ER membranes with half times between 1 and 4 sec. Binding of the components to membranes is diffusion limited and is cargo dependent. It is further regulated by COPI, the p24 family of cargo receptors and microtubule associated motors. Conclusions: Our data suggest that in living cells vesicle formation and coat function are regulated by a so far unappreciated complex network of interactions integrating post-ER traffic and membrane recycling with vesicle formation at the ER level.

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Endothelial Progenitor Cells Friday, July 18, 2003, 14.00
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L6 Transgenic embryonic stem cells in cardiovascular research J. Hescheler, M. Wartenberg, D. Wenzel, B.K.W. Roell, Lu, Y. Xia, F. Do¨nmez, F.C. Ling, H. Acker2, Eugene Kolossov, S. Kazemi, P. Sasse, E. Kolossov, A. Raible, H. Bohlen, W. Bloch, H. Sauer, A. Welz & B.K. Fleischmann Institut fu¨r Neurophysiologie, Robert-Koch-Str. 39, 50931 Ko¨ln, Germany (E-mail: [email protected]) Because of their ability to reproduce the embryological differentiation of nearly all different cellular phenotypes, embryonic stem (ES) cells represent an ideal tool to to study processes of embryogenesis under in vitro conditions, in particular the signaling cascades and genes involved in the functional development (functional genomics) as well as to provide a new source for cellular replacement therapy. We have cultivated ES cells in three dimensional cell aggregates, where they differentiate into derivatives of all three germ layers. (1) Embryonic vascularization comprises different processes such as proliferation, migration, differentiation and tube-formation of endothelial cells. To date little is known about morphogenetic changes of endothelial cells and the molecular mechanisms involved occurring during early stages of embryonic development. We have therefore established stably transfected mouse ES cell lines, where the endothelial-specific platelet endothelial cell adhesion molecule (PECAM) promoter drives the expression of the live reporter enhanced green fluorescent protein (EGFP). This approach enables investigation of morphogenetic changes and related signalling cascades in endothelial cells during early embryonic development. Morphogenetic changes of endothelial cells in the presence of key regulatory molecules fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are monitored employing time lapse microscopy. Three days post plating EGFP positive cells are detected in the ES cell aggregates. At first, clusters of angioblasts are predominant which later develop into elongated network-like structures. VEGF induces proliferation and pronounced sprouting of angioblasts whereas FGF leads to stabilization of preformed endothelial structures. We conclude that the ES cell system in combination with endothelial-specific EGFP expression is a valid tool to investigate early events of vascular differentiation in vitro. VEGF plays an important role for early vessel formation whereas FGF is crucial for endothelial survival. (2) Tumor vascularization is the rate-limiting step for the progression of cancer. Differential steps of tumorinduced angiogenesis were studied by a novel in vitro confrontation-culture of avascular multicellular prostate tumor spheroids and ES cell aggregates grown from ES cells. Vascularization in ES cell aggregates on day 5 of cell culture and was paralleled by downregulation of hypoxia inducible factor 1a (HIF-1a) and VEGF. In parallel a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O2-sensitive microelectrodes. After 24
48 h of confrontation culture, cells positive for PECAM-1 became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days. Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure. Furthermore, tumor vascularization resulted in elevated levels of HIF-1a, VEGF, heat shock protein 27 (HSP27) and P-glycoprotein. Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxiarelated, proangiogenic genes, as well as of HSP27 and P-glycoprotein which are involved in a multidrug resistance (MDR) phenotype. (3) Because of their ability to promote capillarization and angiogenesis ES cells may be usefull for recapillarization after cardiac infarction. This potential is paralleled by their ability to generate cardiomyocytes in vitro for tissue repair. Cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild type mice. Immunological cross reactions were avoided by using the same inbred mouse strain. To allow identification of the transplanted cells transgenic ES cells were used carrying an IRES vector with two cloning sites for EGFP and an antibiotics resistance for selective selection both under the a-MHC promoter. EGFP positive transplanted cardiomyocytes could be easily detected in the native heart at different

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intervals after operation. The cells were found to engraft and differentiate into adult-like cardiomyocytes as confirmed by cross striation after immunostaining with a-actinin. These data were corroborated by patch clamp experiments on isolated EGFP positive cardiomyocytes at different time points after operation. The transplanted cells displayed ventricular action potentials and b-adrenergic- as well as muscarinic regulation. When survival was investigated in a large colony of transplanted- (n ¼ 99) and control mice (n ¼ 36) where NaCl instead of transgenic cardiomyocytes were injected, the control group had an almost double mortality rate. Our data show engraftment and differentiation of embryonic cardiomyocytes after transplantation into cryoinfarcted areas of heart.

L7 Endothelial progenitors: Vehicles for tumor therapy? Jiwu Wei1, S. Blum2, Marcus Unger1, M. Lamparter2, Gergely Jarmy1, Albert Geishauser2, Gordon Chan3, Klaus-Dieter Fischer3, Dirk Rattat4, Klaus-Michael Debatin1, Antonis K. Hatzopoulos2 & Christian Beltinger1 1

Children’s University Hospital, Ulm, Germany; 2GSF-National Research Center for Environment and Health, Munich, Germany; 3Department of Physiological Chemistry, University of Ulm, Germany; 4Department of Nuclear Medicine, University of Ulm, Germany (E-mail: [email protected]) Recruitment of endothelial progenitors is pivotal for the growth and metastasis of tumors. We reasoned that embryonic EPCs (eEPCs), which are easy to expand and to manipulate, would home to tumors and may thus constitute novel vehicles for tumorcytotoxic payloads. Murine eEPCs could be transplanted across allogeneic barriers and remained functional in this setting. They specifically homed to tumors depending on vascularity and, presumably, oxygenation of the tumors. Preliminary in vivo data show that eEPCs engineered to produce a cytotoxic drug may have potential for tumor therapy.

L8 Differentiation of human adult endothelial progenitor cells into functionally active cardiomyocytes Cornel Badorff1, Ralf P. Brandes2, Ru¨diger Popp2, Stefan Rupp1, Masamichi Koyanagi1, Carmen Urbich1, Alexandra Aicher1, Ingrid Fleming2, Rudi Busse2, Andreas M. Zeiher1 & Stefanie Dimmeler1 1

Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany; 2Institute for Cardiovascular Physiology, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany (E-mail: [email protected])

In addition to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Progenitor cells can differentiate into various lineages under appropriate circumstances. The differentiation potential of human adult endothelial progenitor cells (EPCs) into cardiomyocytes is yet unknown. EPCs were isolated from peripheral blood mononuclear cells of healthy adults or coronary artery disease (CAD) patients by cultivating with endothelial cell medium and growth factors. After 3 days, >95% of adherent cells were functionally and phenotypically EPCs. Dil-LDL-labeled EPCs were then co-cultivated with rat cardiomyocytes for 6 days to stimulate EPC differentiation into cardiomyocytes. This co-culture resulted in significant increases of EPC cell length and size to a cardiomyocyte-like morphology. Biochemically, 9.94%  1.39% and 5.04%  1.09% of EPCs from healthy adults (n ¼ 15) or CAD patients (n ¼ 14, P < 0.01 vs. healthy adults), respectively expressed a-sarcomeric actinin as measured by flow cytometry. Immunocytochemistry showed that human EPCs expressed a-sarcomeric actinin, cardiac troponin I (both partly with sarcomeric organization) and ANP. Fluo four-imaging demonstrated calcium transients synchronized with adjacent rat cardiomyocytes in differentiated human EPCs. Individual differentiated human EPCs contracted with adjacent rat cardiomyocytes. Single cell microinjection of lucifer yellow with calcein-AM labeling of

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cardiomyocytes demonstrated gap junctional communication between 51%  7% of EPCs (16 h after labeling, n ¼ 4) and cardiomyocytes. EPC differentiation into cardiomyocytes was not reproduced by conditioned medium, but by co-culture with paraformaldehyde-fixed cardiomyocytes. EPCs from healthy volunteers and CAD patients can differentiate in vitro into functionally active cardiomyocytes, when co-cultivated with rat cardiomyocytes. Cell-to-cell contact, but not cellular fusion mediates EPC differentiation into cardiomyocytes. The therapeutic use of autologous EPCs may aid cardiomyocyte regeneration in patients with ischemic heart disease.

L9 Role of embryonal EPCs in attenuating postischemic reperfusion injury in mice hearts Jan Horstkotte, Christian Kupatt, Alexander Deten, Sabine Blum, Peter Boekstegers & Antonis Hatzopoulos Medizinische Klinik I, Klinikum Großhadern & Ha¨matologikum GSF Munich, Carl-Ludwig Institute of Physiology, Leipzig, Germany (E-mail: [email protected]) Recent reports have demonstrated a beneficial role of adult endothelial progenitor cells (EPCs) after myocardial ischemia and reperfusion. Since adult EPC abundancy after an acute ischemic insult might pose a logistic problem, we investigated the potential of embryonal EPCs to antagonize ischemia/reperfusion injury in mice. Moreover, we investigated mechanisms of PMN and EPC recruitment, since antagonism of PMN recruitment may be applied to avoid reperfusion injury. Methods: Mice (n ¼ 6 per group, wildtype or ICAM-1 -/-) were anesthetized and subjected to LAD-ligation for 20 min. With the onset of reperfusion, 2 · 105 Di-I labeled EPCs were infused into the external jugular vein for 15 min. Thereafter, hearts were arrested and analyzed by fluorescence microscopy for cell recruitment. In parallel experiments (n ¼ 6), circulating leukocytes were stained with rhodamine-6-G and leukocyte adhesion was studied after 15 min of reperfusion. In a chronic model of ischemia (1 h) and reperfusion (14d) we investigated the influence of EPC- and PMN recruitment on systolic myocardial function (left ventricular developed pressure, LVDP) and development of heart failure (LV end-diastolic pressure, LVEDP). Results: Compared to non-ischemic control hearts, ischemia and reperfusion (I/R) induced a significant increase in both, EPC recruitment (10.8  1.8 vs. 1.5  0.4/field) and PMN adhesion (55  5 vs. 8  2/field). ICAM-1 deficiency, however, reduced PMN-recruitment (17  2/field) and not EPC recruitment (8.8  0.8/field). In the chronic model, I/R induced a significant loss of systolic function (LVDP 81  5 vs. 112  4 mm Hg in sham-operated animals), which was attenuated by EPC infusion (2 · 105 cells 24 h after ischemia, LVDP 95  4 mm Hg). Interestingly, ICAM-1 deficient mice also displayed an improvement of LVDP (93  4 mm Hg), which was further extended after EPC-infusion (102  8 mm Hg). LVEDP increased after I/R (7.3  1.1 vs. 4.2  0.3 mm Hg). Either EPC-infusion or ICAM-1 deficiency did not change the elevation of LVEDP (6.8  1.3 mm Hg and 7.1  1.5 mm Hg, respectively). Infusion of EPCs in ICAM-1 deficient mice, however, reduced LVEDP to 4.3  0.8 mm Hg. We conclude that postischemic EPC recruitment does not depend on endothelial ICAM-1 expression, in contrast to postischemic PMN-adhesion. Moreover, ICAM-1 absence and EPC-infusion exert additive cardioprotective effects in a chronic model of ischemia and reperfusion.

L10 Impaired differentiation and adhesion of endothelial progenitor cells under diabetic conditions H.C. de Boer1, C.J.M. Loomans1, E.J.P. de Koning1,2, M.C. Verhaar1, T.J. Rabelink1 & A.J. van Zonneveld1 1

Department of Vascular Medicine, University Medical Center, Utrecht, The Netherlands; 2Department of Diabetology, University Medical Center, Utrecht, The Netherlands (E-mail: [email protected])

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Background: Diabetes mellitus (DM) is a major risk factor for cardiovascular disease and is frequently associated with impaired collateral formation and neovascularization. Recently, it has become clear that neovascularization involves the recruitment of endothelial progenitor cells (EPC), attachment of these cells to endothelium-lined vessels (cell
cell interaction) followed by extension into the interstitium (cell-matrix interaction). Here, we investigated the number of EPC that were obtained in vitro from diabetic- and control Mononuclear cells (MNC) and the adhesion of these cells to human umbilical vein endothelial cells (HUVEC) under flow. Furthermore, we determined the effect of diabetes-associated glycation of endothelial cell matrix (ECM) proteins on the differentiation potential of MNC into EPC. Materials and methods: MNC were isolated from peripheral blood of type 1 DM patients and age-matched healthy volunteers, layered on gelatin and cultured in EPC differentiation medium for four days. In addition, we compared the differentiation of MNC to EPC on gelatin, on isolated ECM from HUVEC and on glycated ECM (incubated for 7 days with 0.5 M glucose-6-phosphate). Adhesion of EPC to TNF-a stimulated HUVEC was tested under flow (1 dyne/cm2). Results: We observed 40% reduction in the number of EPC in type 1 DM patients (n ¼ 9) compared to healthy volunteers (n ¼ 10). This reduction was inversely correlated with patient levels of hemoglobin A1C. Moreover, the adhesion of EPC to TNFa-stimulated HUVEC under flow was reduced. Differentiation of MNC into EPC was dramatically impaired on glycated ECM as compared to gelatin and non-glycated ECM. This reduction was inversely correlated with the degree of glycation. Conclusions: Our data suggest that the diabetic environment hampers the potential of MNC to differentiate into EPC. This impairment is observed at different levels of EPC recruitment, which ultimately may contribute to vascular complications in type I DM.

L11 Impaired expression of MMP-1 in endothelial precursor cells and HUVEC of infants born to HIV-1-infected women Anita Jo¨rg, Eric Guenzi, Kristin To¨polt, Thomas Grimm, Clara Lubeseder-Martellato, Peter Hutzler, Elenore Samson, Thomas Grubert, Robert Oostendorp, Ulrich Keller & Michael Stu¨rzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, 85764 Neuherberg, Germany (E-mail: [email protected]) Background: Infants born to HIV-1-infected women show an increased rate of cardiovascular diseases, even if the infants themselves are HIV-1-negative. In order to investigate if these abnormalities may be caused by malfunction of the endothelial cells we compared the gene expression and biological features of differentiated human umbilical vein endothelial cells (HUVEC) and endothelial precursor cells (EPC) from HIV-1-negative infants born to both healthy and HIV-1-infected women. Materials and methods: EPC were enriched via as CD34+ selection from cord blood using immunomagnetic techniques. EPC adhered and formed colonies after 4
5 days in culture. HUVEC were obtained from umbilical cords of the same infants by collagenase digestion of the interior umbilical vein. Expression profiles were investigated using cDNA macroarray analysis and confirmed by Western blot. Results: Long-term cultures of all EPC differentiated in vitro into endothelial cells expressing Tie-1, PECAM-1, Flk-1, VCAM-1, ICAM-1, E-selectin, CD105 and von Willebrand factor and incorporated acetylated low density lipoprotein. These cells did not differ from HUVEC in their growth characteristics. In addition, both EPC and HUVEC formed capillary-like structures in Matrigel. Interestingly in EPC and HUVEC from HIV-1-negative infants born to HIV-1-infected women the expression of matrix metalloprotease-1 (MMP-1) RNA and protein was abolished or significantly reduced. Conclusion: This is the first report of vertical transmission of a cell biological effect related to HIV-1 independent of the infection with the virus itself. Impaired expression of MMP-1 in EPC and HUVEC from infants of HIV-1-infected mothers may contribute to cardiovascular abnormalities in these children.

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The Endothelium in Development and Angiogenesis I Friday, July 18, 2003, 17.00
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L12 Role of VEGF and endothelial HIFS during embryonic angiogenesis Georg Breier1, Alexander Licht1, Marcel Groot1, Sabine Raab1, Regina Heidenreich1, Judith Schmitz1, Hans-Peter Hammes2, Gerd Elvert3 & Ingo Flamme3 1

Max-Planck-Institut fu¨r physiologische und klinische Forschung, Bad Nauheim, Germany; 2Universita¨tsklinikum Mannheim/Heidelberg, Germany; 3Zentrum fu¨r Molekulare Medizin der Universita¨t Ko¨ln, Germany (E-mail: [email protected]) The development of the vascular system is controlled by endothelial cell specific growth factors and receptor tyrosine kinases. Vascular endothelial growth factor (VEGF) and the high affinity VEGF receptor-2 (VEGFR-2) are essential for the differentiation of the endothelial cell lineage in the mouse. We use conditional gene targeting approaches to study the role of VEGF during brain development. VEGF inactivation in the neural tube led to impaired vascular sprouting and vessel guidance in the developing brain, resulting in brain and skull malformation and perinatal lethality. Heterozygous VEGF-deficient mice lacked the typical oxygen-induced pathological retinal blood vessel proliferation. The expression of VEGF and the VEGF receptors during angiogenesis is controlled by hypoxia-inducible factors (HIF). Analysis of mouse VEGFR-2 gene regulatory elements sufficient for vascular-specific expression in transgenic mouse embryos showed that HIF-2, but not its close relative HIF-1, regulates the expression of VEGFR-2. HIF-2 and ets-1 activated the mouse VEGFR-2 promoter in a cooperative manner and interacted physically. Experiments in transgenic mice that specifically target endothelial HIFs revealed that these factors are essential for the development of the embryonic vascular system.

L13 Growth factors and gene programs for angiogenesis vs. lymphangiogenesis Kari Alitalo & collaborators Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute and Biomedicum Helsinki, P.O.B. 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland (E-mail: [email protected]) Angiogenesis and permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two receptors VEGFR-1 and VEGFR-2. The VEGFR-3 receptor does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. We have found that homozygous VEGFR-3 targeted mice die around midgestation due to failure of cardiovascular development. We have also purified and cloned the VEGFR-3 ligand, VEGF-C. Transgenic mice expressing VEGF-C show evidence of lymphangiogenesis. The proteolytically processed form of VEGF-C binds also VEGFR-2 and is angiogenic. VEGF-D is closely related to VEGF-C, similarly processed and binds to the same receptors. Thus, VEGF-C and VEGF-D appear to be both angiogenic and lymphangiogenic growth factors. Using a novel non-random DNA shuffling method, we have created a library of mosaic molecules starting from the VEGF and the VEGF-C. Mosaic VEGFs were screened for their receptor binding patterns. Some

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VEGF
VEGF-C mosaic molecules with novel receptor binding patterns were obtained; notably molecules that bind all three VEGF receptors. Based on the receptor binding profiles of 400 mosaic molecules and their peptide composition, we have identified receptor specificity determinants and propose a general mechanism of how receptor specificity is achieved within the VEGF family. We have further tested a panel of mosaic molecules in vivo in order to characterize the contribution of individual VEGF receptors for angiogenesis and lymphangiogenesis. We have adapted the mouse corneal angiogenesis assay to study potential lymphangiogenic factors and inhibitors. Immunohistochemical analysis using lymphatic endothelial markers showed that VEGF-C induces lymphatic as well as blood vessel growth in the cornea. By contrast, VEGF induced angiogenesis but not lymphangiogenesis. Interestingly, fibroblast growth factor-2 (FGF-2) stimulated both lymphangiogenesis and angiogenesis. FGF-2 up-regulated VEGF-C expression in vascular endothelial and perivascular cells. Furthermore, administration of blocking anti-VEGFR-3 antibodies inhibited the FGF-2-induced lymphangiogenesis. These findings indicate that VEGFR-3 can mediate lymphangiogenesis induced by other growth factors. As increased expression of FGF-2 and VEGF-C have been associated with lymphatic metastasis (see below), these results may provide a strategy for the inhibition of lymphatic metastasis in cancer therapy. We have targeted the Vegfc gene by replacing its first exon with the b-galactosidase marker gene. Haploinsufficiency or lack of VEGF-C resulted in defects in the lymphatic vasculature in mice. Our findings demonstrate that the Vegfc deficient mice can be used to study potential therapeutic agents for lymphedema or lymphatic dysfunction. Primary human lymphedema, characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding VEGFR-3. We have described a mouse model for this disease. As the human patients, the lymphedema mice have swelling of the limbs due to hypoplastic cutaneous lymphatic vessels. Using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors. Most human tumors have their own characteristic way of metastasizing via lymphatic or blood vessels to specific target tissues. The mechanisms determining these characteristics in different tumor types remain poorly understood. In addition, certain tumor types rarely metastasize. In principle, tumor cells can either directly invade pre-existing lymphatic vessels or new lymphatic vessels formed at the tumor periphery by tumor induced lymphangiogenesis. In fact, tumor cells may use some of the same trafficking pathways, chemokines and adhesion molecules as lymphocytes and antigen presenting cells in order to gain access to the lymphatic vessels. While angiogenesis is required for tumor growth it is not yet clear to what extent active lymphangiogenesis occurs in human tumors. However, intratumoral lymphatic vessels have been documented in tumor xenografts overexpressing VEGF-C or VEGF-D. On the other hand, no intra-tumoral lymphatic vessels were detected in mice overexpressing VEGF-C as a transgene in tumor cells. In our studies, VEGF-C overexpression led to lymphangiogenesis, intralymphatic tumor growth and lymph node metastasis in an orthotopic model of human breast carcinoma in immunoincompetent mice. Furthermore, soluble VEGFR-3, which blocks embryonic lymphangiogenesis, blocked lymphatic metastasis in breast and lung cancer models. Besides being involved in metastasis, the lymphatic vessels have a variety of physiological functions in fluid homeostasis, immune surveillance and fat adsorption. We have found that isolated human primary lymphatic and blood vascular endothelial cells (LECs and BECs, respectively) have interesting differences in gene expression relevant for their distinct functions in vivo. Although these phenotypes are stable in vitro and in vivo, overexpression of the homeobox transcription factor Prox-1 in the BECs was capable of inducing LEC-specific gene transcription in the BECs, and, surprisingly, Prox-1 suppressed the expression of about 40% of the BEC-specific genes. Prox-1 did not have global effects on the expression of LEC-specific genes in other cell types, except that it up-regulated cyclin E1 and E2 mRNAs and activated the cycE promoter in a variety of cell types. Lymphatic development has been previously shown to be arrested in Prox-1 knockout embryos by Wigle and Oliver. Our data and those of Oliver and Detmar suggest that Prox-1 acts as a cell proliferation inducer and as a fate determination factor for the LECs. Furthermore, the data provide important insights into the phenotypic diversity of endothelial cells and into the possibility of transcriptional reproprogramming of differentiated endothelial cells. Veikkola et al., Cancer Res. 60: 203
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L14 Angiogenesis and lymphangiogenesis in tumor progression and inflammation Michael Detmar Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA (E-mail: [email protected]) To directly characterize the biological role and the in vivo molecular control of angiogenesis vs. lymphangiogenesis in inflammation and multi-step tumor progression, we studied transgenic mice overexpressing the angiogenic factors VEGF and PlGF or the angiogenesis inhibitors thrombospondin-1 (TSP-1) and TSP-2, and mice deficient of PlGF or TSP-2. We found that the ‘angiogenic switch’ during multi-step epithelial carcinogenesis already occurred at the early steps of pre-malignant tumor formation, and that genetic overexpression of angiogenesis inhibitors or deficiency of the angiogenic factor PlGF similarly resulted in delayed and reduced early, pre-malignant tumorigenesis, whereas deficiency of TSP-2 or increased levels of VEGF led to enhanced development of premalignant lesions. However, the capacity of premalignant epithelial cells to form malignant tumors (malignant conversion) was not influenced by genetic modulation of angiogenesis. Our recent xenotransplant studies revealed that tumors, through secretion of VEGF-C, can actively induce lymphangiogenesis and thereby promote lymphatic cancer metastasis. Peri- and intratumoral lymphatic vessels were also found in slowly growing, orthotopic squamous cell carcinomas, as evaluated by LYVE-1/Prox1 double stains. Importantly, we found that
in human malignant melanomas
the degree of tumor lymphangiogenesis can serve as a novel prognostic indicator for the metastatic risk and for overall patient survival. Our recent studies indicate that enhanced expression of VEGF also predisposes to chronic cutaneous inflammation that clinically and histologically resembles human psoriasis. VEGF transgenic mice are unable to downregulate experimentally induced inflammation and also display enhanced lymphangiogenesis. Ongoing in vitro studies in lymphatic vs. blood vascular endothelium are aimed at elucidating the (direct or indirect) mechanisms of VEGF-mediated lymphangiogenesis.

L15 Protein complexes transducing ephrinB reverse signaling Stefan Weinges, Ru¨diger Klein & Amparo Palmer MPI of Neurobiology, 82152 Martinsried, Germany (E-mail: [email protected]) EphrinB ligands are important regulators of morphogenic processes such as axon guidance and angiogenesis. The SH2/SH3 adaptor protein Grb4 as well as the PDZ domain-containing glutamate receptor-interacting protein 1 (GRIP1) are both recruited to ephrinB ligands and represent potential targets for the assembly of multiprotein signaling complexes downstream of these ligands. In order to find novel interaction partners for Grb4 and GRIP1 and to subsequently evaluate the relevance of these complexes in ephrin B reverse signaling, we are currently employing the TAP (tandem affinity purification) technology, which allows us to do rapid purification of protein complexes under native conditions. A first TAP purification has revealed a set of interactions with proteins functionally related to the cytoskeleton, an expected target for ephrin/Eph signaling. Novel identified proteins are then validated for in vivo interaction and analyzed for their role in the ephrinB reverse signaling in an in vitro functional assay for migration and invasion of endothelial precursor cells. We are also applying the TAP technology in vivo. Transgenic mice expressing GRIP1-TAP have been generated. This approach allows us to: (a) use an in vivo model; (b) purify complexes from their natural environment, and (c) compare complexes isolated from different tissues and at different developmental stages.

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L16 VEGF withdrawal induces vascular remodeling and pruning via intussusception Valentin Djonov Institute of Anatomy, Bern, Switzerland (E-mail: [email protected]) Once the capillary plexuses have been formed and perfused, they undergo a process of intensive remodelling including formation of feeding vessels and adaptation of the vascular branching morphology. We used the chicken chorioallantoic membrane (CAM) and the chick kidney demonstrating the effects of VEGF withdrawal. We observed emergence of remodelling and adaptation by formation of transvascular tissue pillars i.e. intussusception at vascular bifurcations (intussusceptive branching remodelling, IBR). IBR occurred by formation of pillars close to a bifurcation angle. The pillars increased in girth until they merged with the connective tissue in the bifurcation angle. The formation of multiple eccentric pillars and their subsequent successive fusions meant that IBR was also extensively involved in vascular pruning, a process that results in reduction of the number of vascular branches. Morphometric investigations demonstrated that IBR resulted in optimization of the branching geometry such that optimality was augmented by minimization of pumping energy and building material. Putatively, sub-maintenance doses of VEGF cause vascular remodelling. Application of fibrin matrices containing VEGF121; 165 on CAM caused a pronounced angiogenic response. Subsequent removal of the matrices resulted in extensive intussusceptive vascular pruning, with a following reduction in the number of small arteries and veins. Local application of VEGF inhibitors, induced similar changes in the CAM. Relation between VEGF withdrawal and intussusceptive pruning was also found in the developing kidney. Disappearance of the pro- and mesonephrons is related to the VEGF down regulation followed by massive intussusceptive pruning. We conclude that intussusception plays a crucial role in capillary network growth and expansion as well as vascular tree remodelling, in response to angiogenic factors and changing hemodynamic conditions.

L17 Shear stress induced elastase-fragmentation of laminin to E8 prevents endothelial cell proliferation and differentiation T. Gloe, C. Klarskov, S. Zahler, F. Kro¨tz, H.Y. Sohn & U. Pohl Institute of Physiology, LMU, Munich, Germany (E-mail: [email protected]) Background: Activated matrix proteases generate matrix-fragments that contribute to vascular remodelling. We investigated whether blood flow associated shear stress contributes to the release of elastase and whether elastase fragments of laminin I influence endothelial cell (EC) proliferation and differentiation. Material and methods: Human umbilical vein EC were subjected to shear stress (20 dyn/cm for 2 h) and conditioned media were tested for elastase activity. Western blots of matrix proteins were performed to identify proteolytic laminin fragments. In proliferation assays EC were plated on laminin I, or its fragments P1 or E8. Proliferation was induced by bFGF (3 ng/ml) and measured with MTT. Apoptosis was monitored by cell surface binding to annexin V and free histone ELISA. Oxygen radical production was quantified by cytochrome reduction and DCF florescence. Angiogenesis assays (sprouting) were performed using bFGF stimulated aortic rings from hamster embedded in Matrigel, which were in part supplemented with laminin fragments. Results: Shear stress enhanced elastase activity in conditioned media from 1  0.03 mU/ml in static controls to 16  0.7 mU/ml in shear stress cells (n ¼ 8). This went along with an increased occurrence of the laminin I fragment E8 within the matrix. Seeding EC on purified E8 resulted in a reduced proliferation (P < 0.05, n ¼ 8). In parallel to these findings, EC that were grown on E8 exhibited a 2-fold higher apoptosis rate (n ¼ 4, P < 0.05) as well as an increased O2-production (1.1  0.5 nmol/min on laminin, 2.3  1 nmol/min on E8). Aortic ring

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sprouting was significantly inhibited by E8. This inhibition was revoked by application of radical scavengers SOD or catalase. Conclusion: Upon shear stress EC release elastase, which degrades laminin I into fragments amongst them E8. In contrast to intact laminin I and its fragment P1, E8 inhibits of EC proliferation, induces EC apoptosis, oxygen radical production, and inhibits angiogenesis. These results indicate that during shear stress the matrix is altered post synthesis, a mechanism which might be part of a feed back loop to limit shear stress induced proliferation and differentiation of EC in adaptive vascular remodelling to increased flow.

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The Endothelium in Development and Angiogenesis II Saturday, July 19, 2003, 08.30
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L18 Molecular analysis of the angiopoietin/tie-2 and ephrin/eph ligand receptor systems during angiogenesis Hellmut Augustin University of Freiburg, Breisacherstr. 117, 79106 Freiburg, Germany (E-mail: [email protected])

Abstract not available

L19 Hypoxia signaling and angiogenesis: In vivo interplay between oxygen sensors Edurne Berra, Emmanuel Benizri, Fre´de´ric Dayan, Amandine Ginouve`s, Nathalie Mazure, Danie`le Roux & Jacques Pouysse´gur Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543, Centre Antoine Lacassagne, 33 Avenue Valombrose, 06189, Nice, France (E-mail: [email protected]) Angiogenic factors modulate basic endothelial cell functions such as migration and proliferation, but how these factors are regulated in response to various nutritional stresses, at a molecular level, is not yet fully understood. A remarkable signaling system devised for rapid adaptation to and survival in a low oxygen environment (hypoxia) has been conserved throughout evolution. The hypoxia inducible transcription factor HIF, found in worms, flies and vertebrates is central to this adaptation and as such, hif-1 represents a ‘master’ gene in oxygen homeostasis. HIF-1 is a transcriptional complex that plays a pivotal role in cellular adaptation to low oxygen availability, inducing many genes including VEGF, EPO, GLUT1 and other essential glycolytic enzymes. In the presence of oxygen, the HIF-a subunits are targeted for destruction by proline hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor protein (pVHL). Three mammalian HIF prolyl-hydroxylases (PHD1, 2, 3), homologous to the HIF prolyl-hydroxylase of C. elegans (EGL-9), were recently identified and shown, at least in vitro, to down regulate HIF-a subunits. These enzymes, together with the HIF asparaginyl hydroxylase (FIH), which ‘represses’ the activity of HIF-1a, belong to a large family of non-haem iron oxygenases that require O2 and 2-oxoglutarate for their function. In this presentation we will show that specific ‘silencing’ of HIF prolyl-hydroxylase 2 (PHD2) with short interfering RNAs (siRNA) is sufficient to stabilize and activate HIF-1a, in normoxia, in all the human cells investigated. A remarkable synergy in the HIF-1a activation process is observed in normoxia by ‘co-silencing’ both, PHD2 and FIH. This action measured by a HIF-dependent reporter gene recapitulates a full hypoxic response. Surprisingly, ‘silencing’ of the other two HIF prolyl-hydroxylases, PHD1 and PHD3, had no effect on the stability of HIF-1a either in normoxia or upon re-oxygenation of hypoxic cells. We conclude that PHD2, a cytoplasmic enzyme capable of shuttling between cytoplasm and nucleus, is the critical oxygen sensor setting steady-state levels of HIF-1a, in normoxic conditions. Interestingly, PHD2 is up-regulated by HIF-1 providing an auto-regulatory mechanism driven by the oxygen tension. The stabilization of HIF-1a in norm cells, however, following PHD2 ablation, is progressively ‘desensitized’ within 4
5 days in culture uncovering a more complex mechanism than originally thought. Central to this action appears to be a HIF-dependent activation process, implicating an interplay between oxygen sensors PHD2 and PHD1, a model of which will be presented.

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L20 Intravital fluorescence microscopy of microvascular development in metastades Ian C. MacDonald, Christopher G. Ellis & Hemanth J. Varghese Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada (E-mail: [email protected]) Background: Tumor growth beyond the diffusion distance of metabolites requires a vasculature to supply the needs of the cells. The effectiveness of this vasculature will depend on both its morphology and rate of perfusion. Experimentally, cancer cells injected subcutaneously as a bolus must depend on angiogenesis from vessels recruited from surrounding tissues. In contrast, cells delivered hematogenously to lung or liver will be spread out and by invading throughout the existing vasculature as they begin growth can be supplied by it. However, for continued growth within these metastatic models angiogenesis must also occur. We used intravital fluorescence videomicroscopy (IVFVM) to examine dependence of tumor vascular morphology and perfusion on host tissues. Materials and methods: PAP2 cells (H-ras transfected NIH-3T3 mouse fibroblasts) were injected subcutaneously to form primary tumors, or by intravenous or intraportal routes to form lung and liver metastases. Vascularized tumors were examined by IVFVM using 250 kD FITC-dextran to label plasma. Video sequences were digitally stabilized, and images were produced based on the maximum intensity (to indicate vessel location) and variance (showing non-fluorescent red blood cell motion) for each pixel. Results: Vessels occupied about 50% of the primary tumor volume, and varied widely in diameter, segment length and flow. Volume density was much higher for the lung (70%) but lower for liver (30%) metastases with branching patterns similar to the host tissues. However, flow was only seen in about 60% of the lung tumor vessels, and in less than 30% of the liver tumor vessels with the central portion being very poorly perfused. Conclusions: Digital video sequences captured from IVFVM of FITC labeled plasma in tumor vessels can be processed to form image maps of vessel location based on maximum fluorescence intensity and flow based on the frame to frame variance in intensity at each pixel location. Such images show that while tumor vascular morphology is similar to the surrounding host tissue, the functional microcirculation can be very different.

L21 Specific signals of VEGFR-2 via PKC/EGR-1 and calcineurin/NFAT lead to angiogenic gene induction in endothelial cells H.D. Muller, M. Lucerna, G. Schabbauer, B. Schweighofer, D. Mechtcheriakova, A. Kadl, F. Gruber, B.R. Binder & E. Hofer Department of Vascular Biology, VIRCC, University of Vienna, Austria (E-mail: [email protected]) Background: We have investigated the property of VEGFR-2 to preferentially trigger signals via phospholipase C-gamma, PKC and calcineurin and its relevance for gene induction and angiogenesis. Materials and methods: To investigate the participation of various signalling pathways in VEGF-mediated gene induction we have used Western blotting, real-time PCR and reporter gene assays following overexpression of signalling molecules and transcription factors. Effects on angiogenesis were evaluated using adenoviral overexpression. Results: The data show that in contrast to other growth factors VEGF strongly triggers signals leading via PKC/ MEK/ERK and Ca++/calcineurin to the induction of the transcription factors EGR-1 and NFAT in endothelial cells. It appears that the PKC-epsilon isoform is significantly involved. PKC-epsilon-induced EGR-1 and calcineurininduced NFAT cooperate in gene regulation to induce the tissue factor (TF) gene and some other genes regulated by VEGF. Using TF as a readout for VEGF-induced genes we demonstrate that inhibition of EGR-1

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and NFAT by either overexpression of the specific corepressor NAB2 or by cyclosporin A, respectively, inhibits VEGF induction of the TF gene and protein. Furthermore, a recombinant adenovirus expressing NAB2 inhibited expression of several genes proposed to be important for angiogenesis including VEGFR-1 and prourokinase. In several in vitro and in vivo angiogenesis models, sprouting and tubule formation was reduced by the NAB2 adenovirus supporting an important role of EGR-1. Conclusion: Our data suggest that primarily the PKC-epsilon/EGR-1, but to some extent also the calcineurin/ NFAT pathway, could be promising targets for the therapeutic modulation of angiogenesis.

L22 Cytochrome P450 2C9 induces cyclo-oxygenase-2 (COX-2) expression in endothelial cells U.R. Michaelis, B. Fisslthaler, R. Busse & I. Fleming Institut fu¨r kardiovaskula¨re Physiologie, Klinikum der J.W.Goethe-Universita¨t Frankfurt, Germany (E-mail: [email protected]) The endothelial cytochrome P450 epoxygenase 2C9 (CYP 2C9) plays an important role for vascular homeostasis via metabolising arachidonic acid into epoxyeicosatrienoic acids (EETs). EETs do not only activate Ca2+-dependent K+-channels and protein kinases, they also induce angiogenesis. The different signalling pathways leading to EET-induced angiogenesis are not yet completely understood. COX-2 is known to be involved in angiogenic processes and its expression correlates with the formation of new blood vessels. Therefore we investigated whether CYP 2C9 overexpression modulates the expression of COX-2. To investigate the effect of CYP 2C9 on COX-2 expression, human umbilical vein endothelial cells (HUVEC) were infected with CYP 2C9 sense or antisense adenoviral constructs. Overexpression of CYP 2C9 induced a two-fold increase in COX-2 protein expression, an effect which was accompanied by elevated prostaglandin levels and a two- to threefold increase in COX-2 promoter activity. The CYP 2C9-induced expression of COX-2 was sensitive to the specific CYP 2C9 inhibitor sulfaphenazole. Furthermore, the protein kinase A inhibitor KT5720 attenuated the CYP 2C9-induced increase in COX-2 promoter activity and protein expression. Moreover, exogenous application of 11,12-EET led to an increase in COX-2 expression in endothelial cells. Stimulation with 11,12-EET markedly increased intracellular cyclic AMP levels and activated and therefore induced DNA-binding of the cyclic AMPresponse element-binding protein (CREB). Taken together, these data show, that CYP 2C9 induces COX-2 expression in endothelial cells via a cyclic AMP-dependent pathway, a mechanism which probably contributes to the CYP 2C9-induced angiogenic response.

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The Endothelium in Atherosclerosis Saturday, July 19, 2003, 10.50
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L23 Inflammation induced by oxidized lipids: A role in atherosclerosis Norbert Leitinger Department of Vascular Biology and Thrombosis Research, University Wien, Schwarzspanierstr. 17, 1090 Wien, Austria (E-mail: [email protected]) There is increasing evidence that lipid oxidation products may play an important role in the pathogenesis of atherosclerosis as well as other chronic inflammatory diseases. Oxidized lipids activate cells of the vascular wall and blood cells to induce expression of inflammatory genes. Moreover, lipid oxidation products stimulate endothelial cells to specifically bind monocytes, but not neutrophils, a hallmark of chronic inflammatory diseases. We could show that different lipid oxidation products such as oxidized phospholipids, oxidized cholesteryl esters and isoprostanes all were capable of inducing specific monocyte adhesion to endothelial cells. Activation of the MAP-kinase signaling cascade, rather than the classical NF-jB pathway, was shown to be important for this effect. Furthermore, tissue factor (TF) expression induced by oxidized phospholipids (OxPAPC) was mediated by the PKC/MEK/ERK/EGR-1 and Ca++/calcineurin/NFAT pathways, not involving the NF-jB pathway. In contrast to the upregulation of proinfiammatory genes, oxidized phospholipids were also shown to induce the expression of protective enzymes such as HO-l, which is the rate-limiting enzyme in heme-catabolism and has antioxidative capacity. Recent data suggest that oxidized phospholipids inhibit up-regulation of E-selectin, which is characteristic of acute inflammation. Moreover, there is evidence that LPS-induced NFjB-mediated inflammation is down regulated by OxPAPC, leading to the hypothesis that lipid oxidation products may promote the shift from an acute inflammatory response to a chronic state. The inhibitory effect could not be overcome by raising LPS concentration and could not be mimicked by other lipids. In LPS-injected mice, oxidized lipids inhibited the induction of inflammatory adhesion molecules, extravasation of blood cells and protected mice from lethal endotoxin shock. Furthermore, our data demonstrate that oxidized lipids inhibit effects of LPS through a noncompetitive mechanism, blocking the interaction of LPS with LBP and CD14. We hypothesize that oxidized phospholipids, which accumulate at sites of acute bacterial inflammation due to high concentrations of reactive oxygen species generated by neutrophils, can function as a negative feedback to down-regulate the process of acute inflammation. Furthermore, identified chemical structures capable of inhibiting endotoxin can be used for the development of new drugs for sepsis treatment. Thus, identification of mechanisms and signaling pathways induced by oxidized lipids that modulate inflammatory response in the vascular wall will lead to novel strategies of therapeutic intervention in chronic inflammatory diseases.

L24 The critical role of L-arginine in endothelial cell survival during oxidative stress Christoph V. Suschek1, Oliver Schnorr1, Karsten Hemmrich1, Olivier Aust2, Lars-Oliver Klotz2, Helmut Sies2 & Victoria Kolb-Bachofen1 1

Research Group Immunobiology, Heinrich-Heine-University Duesseldorf, Germany; 2Institute of Physiological Chemistry I and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-University Duesseldorf, Germany (E-mail: [email protected])

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Background: Oxidative damage of vascular endothelium represents an important initiation step in the development of atherosclerosis. Recently, we had reported that high-output synthesis of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) serves a powerful protective activity from various stresses in endothelial cells. As iNOS activity critically depends on the availability of its substrate L-arginine, the present study aims at elucidating iNOS-mediated effects on H2O2-induced apoptosis of cytokine activated rat aortic endothelial cells (AEC) subject to medium L-arginine concentrations. Methods: Primary rat endothelial cells were obtained by outgrowth from aortic rings and each batch was screened for purity by monitoring endothelial antigen expression. Cells were cultured in RPMI 1640 without arginine, which was added at concentrations indicated. Results: In cytokine activated AEC, iNOS activity was found to be half-maximal at 60 lM arginine, which represents the normal serum level in rats and also in humans. Maximal activity is seen at and above 200 lM arginine. Activated cells grown in the absence of arginine with minimal iNOS activity are highly sensitive towards H2O2-induced apoptosis and increases in medium arginine concentrations result in increased cell survival. Moreover, competition experiments show that iNOS activity is completely dependent on cationic amino acid transporter (CAT)-mediated arginine uptake. We also find that the arginine-dependent protection includes inhibition of endothelial lipid peroxidation and increases in the expression of vasoprotective stress response genes. Conclusions: Our data demonstrate that arginine concentrations corresponding to physiological serum levels do not allow for optimal endothelial iNOS activity. Thus, decreases in systemic or local arginine concentrations will impair the endothelial iNOSmediated stress response and increase endothelial dysfunction.

L25 Alpha-tocopherol and its water-soluble derivative trolox potentiate endothelial no synthesis via different mechanisms R. Heller1, M. Hecker1, G. Werner-Felmayer2 & E.R. Werner2 1

Institute of Molecular Cell Biology, University of Jena, Germany; 2Institute of Medical Chemistry, University of Innsbruck, Austria (E-mail: [email protected])

Background: Reduced generation of endothelium-derived nitric oxide (NO) leads to vasomotor dysfunction and disordered thromboregulation and has been implicated in the development of atherosclerosis. The activation of endothelial NO synthase (eNOS) is mediated by an increase of intracellular calcium and/or by enzyme phosphorylation at ser1177 and is critically dependent on the availability of its substrate arginine and its cofactor tetrahydrobiopterin (BH4). BH4 has been shown to be chemically stabilized by ascorbic acid. The most common form of vitamin E in human tissues, alpha-tocopherol, seems to be involved in the regulation of vascular homeostasis but the underlying mechanisms are poorly characterized. The present study investigates whether alpha-tocopherol or its short-chain, water-soluble analog trolox affect NO synthesis in endothelial cells (EC). Methods: NO generation was detected after ionomycin stimulation of human umbilical vein EC as formation of citrulline and cGMP (co-product and effector molecule of NO, respectively). The expression of eNOS and phosphorylated eNOS in cell lysates or cellular subfractions was analyzed by Western blotting using specific anti-eNOS or anti-phospho-eNOS (ser1177) antibodies. The concentration of BH4 was calculated from biopterin levels measured with HPLC in EC extracts oxidized under acidic or alkaline conditions. GTP cyclohydrolase I (GTP-CH), the key enzyme of BH4 synthesis, was analyzed by quantitative RT-PCR. Results: The ionomycin-induced eNOS activation was paralleled by an increased phosphorylation at ser1177 which was prevented by calmodulin-dependent kinase II-inhibition. Preincubation of cells with alpha-tocopherol or trolox (10
200 lM, 24 h) potentiated ionomycin-stimulated citrulline- and cGMP-formation up to 1.5-fold or 2.2-fold, respectively, without changing the expression or the subcellular distribution of eNOS. The effect of alpha-tocopherol on NO formation was amplified by coincubation with ascorbic acid (100 lM, 24 h) whereas the effect of trolox was abolished by ascorbate.Trolox but not alpha-tocopherol led to a 2-fold increase of endothelial BH4 concentration which correlated with a decrease of BH4 oxidation whereas GTP-CH expression and BH4 synthesis were not altered. On the contrary, alpha-tocopherol but not trolox increased and prolonged ionomycininduced eNOS phosphorylation at ser1177 which was augmented by ascorbate. Conclusions: Our data describe a new vasoprotective function of alpha-tocopherol, i.e. the potentiation of eNOS activation, whereas its water-soluble analog trolox like ascorbate protects the eNOS cofactor BH4 from oxidation. The stabilizing effect of trolox on BH4 is effective only in cells which are not saturated with ascorbate.

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Ascorbic acid amplifies the effect of alpha-tocopherol on ser1177 phosphorylation possibly by regenerating the compound and both substances act synergistically in providing optimal conditions for endothelial NO synthesis.

L26 Chemokines and adhesion molecules in atherogenic monocyte recruitment Christian Weber Kardiovaskula¨re Molekularbiologie, RWTH Aachen, Germany (E-mail: [email protected]) Chemokines expressed on atherosclerotic endothelium or delivered by platelets have been implicated in monocyte recruitment during atherogenesis and restenosis. When presented by early atherosclerotic lesions, the endothelial chemokine KC (CXCL1) and the platelet-derived chemokine RANTES (CCL5) but not MCP-1 (CCL2) triggered integrin-mediated monocyte arrest, indicating a functional specialization in atherogenic recruitment. Activated platelets supported monocyte arrest on vascular cell adhesion molecule-1 (VCAM-1) expressed by inflamed or atherosclerotic endothelium, and promoted formation of atherosclerotic lesions in apoE-/- mice. This role of activated platelets in atherosclerosis was largely attributable to platelet P-selectin-mediated delivery of chemokines to monocytes or the vessel wall. In particular, RANTES deposition required platelet P-selectin, and was involved in wire-induced intimal hyperplasia in apoE-/- mice. The chemokine-related cytokine macrophage inhibitory factor (MIF) is expressed in atherosclerotic endothelium but upregulated in endothelial and neointimal cells in the shoulder region of plaques after arterial injury in apoE-/- mice. Neutralization of MIF reduced macrophage and foam cell content, while increasing smooth muscle cell and collagen content in the neointima. Deposition of MIF on endothelium enhanced monocyte arrest, while recruitment on endothelium stimulated with oxidized LDL was mediated by endothelial MIF. Inhibition of MIF thus shifted the cellular plaque composition towards a stabilized phenotype. In addition to the known role of immunoglobulin family members, e.g. VCAM-1, junctional adhesion molecule-1 was detectable on atherosclerotic endothelium, and may be as crucially involved in transendothelial diapedesis of monocytes, as for inflamed endothelium. Understanding the molecular actions of chemokines and adhesion molecules in atherosclerosis may help to establish novel strategies for their therapeutic targeting.

L27 Enhanced UPAR expression in atherosclerotic lesions of APO E-deficient mice is associated with a decrease in b3-endonexin and enhanced NFjB activity Felicitas Besta1, Steffen Massberg, Korbinian Brand2 & Meinrad Gawaz3 1

Deutsches Herzzentrum Mu¨nchen, TU Mu¨nchen, Germany; 2Institut fu¨r Klinische Chemie, Klinikum rechts der Isar, TU Mu¨nchen, Germany; 3Deutsches Herzzentrum Mu¨nchen, TU Mu¨nchen, Germany (E-mail: besta@ dhm.mhn.de) Background: Proteolysis of extracellular matrix components is required for cell migration occurring in atherosclerotic lesion formation. In the present study, gene expression of the protease-receptor uPAR and underlying mechanisms were analyzed during the development of atherosclerosis in the aorta of apolipoprotein E-deficient mice (apoE-/-). Material and methods and results: Immunohistochemical staining revealed a significant increase of uPAR expression in the atherosclerotic tissue with advancing plaque-dimension. ELISA and semiquantitative real-time polymerase chain reaction exhibited an enhanced uPAR-expression in aortic tissue of apoE-/- mice compared to wildtype mice. As uPAR gene transcription involves the transcription factor NFjB, we analyzed nuclear NFjBactivity in vascular tissue of apoE-deficient mice. Congruent to uPAR, we could detect an increase in NFjB-

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activity by EMSA. Parallel we found enhanced IjB-a levels and IKKinase activity, which underlines the chronical inflammatory component of the disease. Recently we reported that b3-endonexin, a protein that modulates b3integrins, regulates uPAR expression through direct interaction with subunits of the NFjB-complex. Herein we could show that b3-endonexin protein is expressed in aortic tissue of mice. Moreover, in contrast to uPAR respectivly NFjB, the expression of b3-endonexin was reduced in nuclear extracts of advanced atherosclerotic plaques, suggesting that the decrease of the protein is critical in the atherogenetic process. As other NFjB regulated genes like MCP-1 and ICAM-1 were also found to be overexpressed in atherosclerotic tissue we investigated the effect of b3-endonexin on these early inflammatory proteins. In vitro stimulation of endothelial cells by vitronectin, an extracellular matrix component resulted in a significant enhanced NFjB-activity and MCP1 expression. The increase in MCP-1 was notedly reduced in cells that were overexpressing b3-endonexin. These results suggest that b3-endonexin acts as a regulating factor in the integrin-mediated signal transduction and plays an important role in the inflammatory process of atherosclerosis.

L28 Role of platelet-endothelial cell interactions for atherosclerosis* Steffen Massberg1, Ildiko Konrad1, Sabine Gru¨ner1, Iris Mu¨ller1, Tomas Richter2, Michael Lorenz1, Julia Kersting1, Katrin Hemler1, Wolfgang Bergmeier3, Bernhard Nieswandt3 & Meinrad Gawaz1 1

Deutsches Herzzentrum und 1. Medizinische Klinik, Technische Universita¨t, Mu¨nchen; 2Institut fu¨r Pathologie, Technische Universita¨t, Mu¨nchen, 3Rudolf Virchow Center for Experimental Biomedicine, Wu¨rzburg (E-mail: [email protected])

Background: Endothelial dysfunction during the process of atherosclerosis is thought to alter endothelial antiadhesive and antithrombotic capacities. Platelets have been demonstrated to adhere to dysfunctional endothelial cells. Upon adhesion platelets release proinflammatory mediators. In this manner platelet-endothelial cell interactions might play an important role in the process of atherogenesis. However, the contribution of platelets to atherosclerotic lesion formation remains unclear. Materials and results: We used intravital videofluorescence microscopy to determine platelet adhesion dynamics in ApoE-deficient mice of different ages. We show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE-deficient mice prior to the development of manifest atherosclerotic lesions. Platelet-endothelial cell interaction involved both platelet glycoprotein (GP)Iba and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and strictly preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE-deficient mice profoundly reduced leukocyte accumulation in the arterial intima as assessed in vivo by fluorescence microscopy and ex vivo by assessment of CD14 expression. Moreover, inhibition of platelet adhesion attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. Conclusions: These findings establish the platelet as a major player in initiation of the atherogenetic process. *Supported by the GRK 438.

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The Endothelium in Inflammation Saturday, July 19, 2003, 14.00
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L29 Molecular mechanisms involved in the control of interendothelial cell contacts Dietmar Vestweber Max Planck-Institute of Vascular Biology and Institute of Cell Biology, ZMBE, University of Mu¨nster, Germany (E-mail: [email protected]) The mechanisms that regulate adhesion between endothelial cells are only poorly understood. It is likely that they are involved in the process of leukocyte extravasation. The junctional adhesion molecules (JAMs) form a recently identified sub-family (Three members known) of the immunoglobulin supergenefamily containing two Igdomains. They are expressed on epithelial and endothelial cells, at least two of them seem to be associated with tight junctions, and to be involved in leukocyte extravasation. We and others have recently identified another two Ig-domain protein, called ESAM, which is endothelial specific, mediates cell aggregation and is closely associated with tight junction markers. Similarities and differences between ESAM and the JAMs will be discussed. Tyrosine phosphorylation of adherens junction components has long been suspected to be involved in the regulation of junction integrity and a correlation between tyrosine phosphorylation of cadherin/catenin complexes and changes in the stability of cell contacts has been well documented. VE-cadherin is the essential adhesion molecule in endothelial adherens junctions. We have found that VE-PTP (vascular endothelial-protein tyrosine phosphatase), an endothelial receptor-type phosphatase co-precipitates with VE-cadherin, but not with b-catenin from cell lysates of transfected COS-7 cells and of endothelial cells. While interactions of other RPTPs with cadherin
catenin complexes are dependent on their cytoplasmic domains, co-precipitation of VE-cadherin and VE-PTP required the most membrane proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor-receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells, transfected with VE-cadherin and VEGFR-2 increased VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VEcadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VEcadherin that associates through an extracellular domain, and reduces tyrosine phosphorylation of VE-cadherin and cell layer permeability independent of its enzymatic activity.

L30 Platelet adhesion to the endothelium: Role for inflammation and microcirculation Meinrad Gawaz 1

Medizinische Klinik, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Germany (E-mail: meinrad. [email protected]) Although the pathophysiological role of platelets in acute thrombotic vessel occlusion in the vicinity of the plaque rupture are well investigated, the significance of platelets for microcirculatory disturbances is less well understood. Experimental results confirm that there is an increased adhesion of activated platelets to the dysfunctional endothelial cell layer. Platelet adhesion to the endothelium involves P-selectin expressed by the

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endothelial surface (mediating initial platelet tethering) and the platelet fibrinogen receptor, which associates with fibrinogen bound to the endothelium and promotes stable platelet arrest. Both, ICAM-1 and the vitronectin receptor are thought to mediate fibrinogen binding and subsequent platelet recruitment to the luminal aspect of endothelial cells. Since platelet adhesion to postischemic endothelial cells involves the interaction between GPIIb-IIIa and fibrinogen bridging to active vitronectin receptors expressed on the endothelial surface, the adhesion of platelets to endothelial cells can be inhibited by antagonists of b3-integrins such as 7E3, RGD, or a vitronectin-specific peptide c(RGDfV). During adhesion platelets release a variety of proinflammatory compounds including interleukin-1b and CD40 ligand that modify the chemotactic and adhesive properties of endothelium and favor leukocyte adhesion and extravasation to the site of inflammation. It is conceivable that the increased adhesion of platelets to endothelial cells, especially in the region of smaller coronary vessels leads to impairment of the microcirculation of diseased tissue that contributes to ischemia. Hence, a targeted pharmacological intervention in the mechanism of platelet-endothelium adhesion could have a favorable influence on the clinical course of reperfusion injury in patients with myocardial infarction or after organ transplantation.

L31 The guanylate binding protein-1 is a tissue and serum marker of inflammatory cytokine-activated endothelial cells Clara Lubeseder-Martellato, Eric Guenzi, Kristin To¨polt, Anita Jo¨rg, Elisabeth Naschberger, Erwin Tschachler, Norbert H. Brockmeyer & Michael Stu¨rzl Deparment of Virus-Induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center of Environment and Health, 85764 Neuherberg, Germany (E-mail: [email protected]) Background: Inflammatory cytokines (IC) such as IFN-a, IFN-c, IL-1a, IL-1b and TNF-a are potent paracrine activators of endothelial cells (EC) in numerous patho-physiological processes, including inflammation, wound healing, tumor angiogenesis and atherosclerosis. GBP-1 is a large GTP-ase that is specifically induced by the above listed IC in EC in vitro. Materials and methods: Standard immunohistochemical and immunofluorescence stainings on paraffin embedded tissues and fixed cells. Detergent extractions of proteins from cell extracts. Immunoprecipitation of proteins from cell culture media. Lactate dehydrogenase activity assay. Development of a three step sandwich ELISA specific for GBP-1: sensitivity 2 ng/ml, intra-assay variability less then 6%. Results: Immunohistochemical studies indicated that GBP-1 expression was clearly up-regulated and strongly EC-associated in skin diseases with an inflammatory component including psoriasis, adverse drug reactions of the skin and Kaposi´s sarcoma as compared to human healthy skin. Moreover, immunoprecipitation experiments showed that GBP-1 is secreted into the cell culture medium of IFN-a- and IFN-c-treated EC, but not of fibroblasts. This result was confirmed by the use of a GBP-1 sandwich ELISA developed in this laboratory. GBP-1 secretion was energy dependent and occurred in the absence of cell death. Preliminary experiments showed that secreted GBP-1 retained its GTP-ase activity. In vivo circulating GBP-1 could be detected in the blood of patients with melanoma under IFN-a therapy and in the blood of patients with inflammatory skin diseases. In addition, serum concentrations of GBP-1 were high in patients at the onset of AIDS and decreased constantly with disease progression. Conclusion: GBP-1 is a novel marker of IC-activation of EC in vivo both at the histological and the serological level. This marker may be helpful to chart the IC activated phenotype of EC in inflammatory diseases, for the early detection of inflammation and to monitor the immunological status of AIDS patients.

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L32 The GTPase function of GBP-1 controls invasiveness and angiogenic capability of endothelial cells through the inhibition of MMP-1 expression Eric Guenzi, Kristin To¨polt, Clara Lubeseder-Martellato, Anita Jo¨rg, Elisabeth Naschberger, Roberto Benelli, Adriana Albini & Michael Stu¨rzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, 85764 Neuherberg, Germany (E-mail: [email protected]) Background: Angiogenesis is an invasive and proliferative process where the coordinated activation of endothelial cells (EC) by inflammatory cytokines (IC) such as IL-1b, TNF-a and IFN-Y and angiogenic growth factors (AGF) such as bFGF and VEGF leads to the formation of new vessels emerging from preexisting ones. We have shown that the major activation pathways of EC during angiogenesis, namely the IC pathway and the AGF pathway, converge in the regulation of the expression of the large GTPase guanylate binding protein-1 (GBP-1). IC induced GBP-1 expression in EC in vitro and in vivo and are potent inhibitors of EC proliferation. In contrast, GBP-1 expression is inhibited by VEGF and bFGF that are potent activators of EC proliferation and invasion. The inhibition of EC proliferation by IC is mediated by the helical domain of GBP-1 and does not require its GTPase function. Materials and methods: Gene expression of HUVEC transduced with retroviral vectors that express a GBP-1 full-length cDNA in sense (S-GBP-1) or antisense (AS-GBP-1) orientation was analysed using cDNA array technology (Atlas, Clontech). Angiogenic capability of transduced HUVEC was analysed in migration and tube formation assays in three dimensional collagen I matrices. Results: We show that the expression of matrix metalloproteinase-1 (MMP-1) is inhibited in S-GBP-1transduced HUVEC. Expression analysis of MMP-1 in HUVEC under IC or AGF stimulation demonstrated that MMP-1 expression is inhibited by IC and that this is mediated by GBP-1. The GTPase activity of GBP-1 was necessary, but not sufficient, for this effect and a GTPase-deficient mutant operated as a dominant inhibitor of wild type GBP-1, suggesting the requirement for oligomerization. This activity of GBP-1 potently inhibited invasiveness and tube forming capability of EC in collagen-I matrices. Conclusion: Invasion of EC into the extracellular matrix is a key feature of angiogenesis. The capability of GBP-1 to inhibit invasion and proliferation of EC establishes this molecule as a key mediator of the IC-induced anti-angiogenic phenotype of EC.

L33 Imaging of haemostatic active Von Willebrand factor multimers on the apical surface of human endothelial cells Rainer Ossig, Hans Oberleithner, Alexej Barg & Stefan W. Schneider Department of Dermatology and Department of Physiology, University Muenster, Germany (E-mail: [email protected]) Von Willebrand factor (vWF) is the major player of the first response of primary haemostasis. Immobilized to the subendothelial cell matrix vWF is exposed after endothelial cell (EC) damage to initiate platelet clotting. Our data indicate that vWF is also released at the luminal surface of EC mediating platelet adhesion. By using atomic force microscopy (AFM) we visualized the luminal surface topography of EC. Submembrane Weibel
Palade bodies (WPB; storage vesicles of vWF) can be imaged as humps characterized by decreased cell membrane stiffness. After cell stimulation (hyperosmolaric solutions or histamine) these docked WPB fuse with the plasma membrane forming large exocytotic pores (d  300 nm). Release of vWF from these pores was visualized by using immunofluorescence after staining the same cell surface with vWF-specific antibodies. Surprisingly, high-

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molecular weight multimers of vWF were found to form elongated fibre-like structures (>200 lm in length) on the EC cell surface. After adding platelet-poor plasma during a 3 min stimulation time cell surface attached platelets were found to be almost exclusively sticking to vWF fibres. Moreover, we confirmed that luminal release and surface attachment of high-molecular weight vWF fibres on EC at the intact human vessel wall. Our data indicate a new concept for a physiological function of vWF to effectively recruite platelets to the intact but stimulated vessel wall surface.

L34 Circulating endothelial cells in bone marrow transplantated patients
a marker of endothelial toxicity? A. Woywodt1, J. Scheer1, L. Hambach2, S. Buchholz2, A. Ganser2, H Haller1, B. Hertenstein2 & M. Haubitz1 1

Abt. Nephrologie, 2Abt. Ha¨matologie/Onkologie, Medizinische Hochschule Hannover, Germany (E-mail: [email protected])

Background: Major complications after allogeneic bone marrow transplantation (BMT) like GvHD, microangiopathic hemolytic anemia, hepatic veno-occlusive disease and capillary leak syndrome were pathophysiologically related to endothelial damage. Conditioning regimens are thought to play an important role in this process. However, data are sparse and contradictory. We have shown recently that circulating endothelial cells (CEC) are a promising marker of endothelial toxicity (Woywodt et al. Hypertension, 2003). Therefore we decided to investigate CEC in patients with BMT. Methods: 31 patients (18 men/13 women, age 22
62 median 45 years; acute leukemia n ¼ 18, chronic myeloid leukemia n ¼ 8, other n ¼ 6) were studied. Conventional conditioning was used in 23 patients (busulfan and cyclophosphamide (Bu/Cy) or total body radiation and cyclophosphamide (TBI/Cy)). Reduced intensity conditioning (RIC) was used in eight patients (fludarabine and busulfan (n ¼ 7) or melphalane (n ¼ 1)). CEC were measured 7 and 1 days before and 7, 14 and 21 days after transplantation. CEC were isolated from peripheral blood by use of Dynabeads coated with antibodies against CD146, were stained for UEA-1 and counted. Results: Patients before BMT had already elevated CEC (median 16, range 8
64 cells/ml) compared to normal controls (median 8/ml; P < 0.01). CEC increased after start of conditioning with maximal CEC of 16-424 (median 52/ml P < 0.05, Mann
Whitney U test). Patients with Bu/Cy (n ¼ 11) showed a significant increase 14 days after BMT (median 36; P < 0.01, paired Wilcoxon test) while in patients with TBI/Cy (n ¼ 12) a much earlier increase after conditioning could be shown (1 day before BMT median 42/ml P < 0.01; 7 days after BMT median 38 P < 0.03; 14 and 21 days after BMT median 36 and 28/ml respectively). In patients with RIC CEC increased 14 days after BMT (median 26/ml; P < 0.05) but were significantly lower compared to conventional Bu/Cy (P < 0.05). Conclusion: CEC are increased in patients before conditioning probably due to cytoto- xic pretreatment. Cells increased further after start of conditioning regimens. Differences in the magnitude and the time point of the increase could be shown between different conditioning regimens. Reduced intensity conditioning led to a lower increase in CEC. CEC proved a interesting new tool to study endothelial toxicity in patients after BMT.

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The Endothelium as a Target of Therapy Saturday, July 19, 2003, 16.15
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L35 Cellular and molecular basis for the selective antiangiogenic effects of frequent low-dose (metronomic) chemotherapy Guido Bocci1, Shan Man1, Jack Lawler2, Francesco Bertolini3 & Robert S. Kerbel1 1

Department of Medical Biophysics, University of Toronto, Toronto, Canada; 2Department of Pathology, Beth Israel Deaconess Medical Center, Boston, USA; 3Division of Hematology-Oncology, European Institute of Oncology, Milan, Italy (E-mail: [email protected]) Many conventional chemotherapeutic drugs have significant antiangiogenic activity in tumors which is detected especially when such drugs are administered on a frequent schedule at doses substantially lower than the maximum tolerated dose (MTD), i.e., ‘metronomic’ therapy. The basis for this is considered to be the presence of dividing endothelial cells present in newly formed tumor blood vessels. Like other normal dividing host cells, these cells appear to be sensitive to most chemotherapeutic drugs. However, damaged or destroyed tumor associated endothelial cells may be rapidly replaced during the long rest periods between standard MTD cycles of chemotherapy, which can be avoided by more frequent (e.g. daily weekly) schedules of drug administration. Because of reduced toxicity, metronomic chemotherapy regimens are particularly well suited for chronic administration and hence integration with newly developed targeted antiangiogenic drugs. Such combinations appear to be much more effective than metronomic chemotherapy or antiangiogenic drug administration alone. Why shortening the breaks between chemotherapy does not also cause an increase in the rate of damage and cell death of other regenerating chemosensitive cells/tissues is unknown, but implies a strongly selective of lowdose chemotherapy for activated endothelial cells. In this regard, several groups, ours included, have shown that endothelial cells are growth inhibited or killed by extraordinarily low concentrations of various chemotherapeutic drugs, in comparison to other cell types. We have recently obtained evidence that this striking sensitivity and selectivity may be secondary to induction in endothelial cells of thrombospondin-1 (TSP-1), a specific endogenous inhibitor of angiogenesis which binds to CD36 receptors on endothelial cells. Thus, some continuous low-dose chemotherapy (cyclophosphamide) regimens lose their antiangiogenic and anti-tumor effects in TSP-1 knockout mice. In addition, we have obtained evidence which shows that metronomic low-dose cyclophosphamide regimens induce a sustained and highly significant drop in the levels and viability of circulating endothelial progenitor cells, whereas MTD regimens of the same drug does not. Whether this is also mediated by TSP-1 is not yet known. Together, our latest results may help explain the antiangiogenic basis and selectivity of metronomic chemotherapy regimens, and why MTD regimens of the same drugs have much less potent antiangiogenic effects and greater host toxicity.

L36 Wadih Arap & Renate Pasqualini Department of Genito-Urinary Medical Oncology, University of Texas, M.D. Anderson Cancer Center, T7.3970 Houston, Texas, USA (E-mails: [email protected], [email protected]) Abstract not available

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L37 Targeting tumor vasculature using specific homing peptides: Pitfalls and prospects Markus Essler Department of Nuclear Medicine, Klinikum rechts der Isar, Ismaningerstr. 22, 81675 Mu¨nchen (E-mail: [email protected]) Tumor homing peptides are effective tools to improve the efficacy of anti-cancer drugs in pre-clinical models of cancer. Here I report the discovery of two peptides specifically homing to the vasculature of breast cancer. A cyclic nonapeptide binds to the vasculature of normal breast tissue, hyperplastic lesions and early breast cancer, but not to metastases or advanced tumors. In contrast a linear pentapeptide homed to breast cancer metastases and advanced tumors, but not to normal breast tissue. Though both peptides are effective in targeting tumor vasculature, they bind tumor blood vessels only in distinct stages of maturation. I also provide evidence that most tumor homing peptides discovered far
including the well characterized RGD4C peptide
are effective only in specific stages of maturation of the vasculature. Hence, the success of targeted cancer therapies using vascular homing peptides will depend on techniques predicting the efficacy of the homing peptides in individual patients, because tumor blood vessel maturation may be different in each patient. Scintigraphic imaging of tumors could be a useful technique for the selection of homing peptides for individualized cancer therapies.

L38 Development of novel angiogenesis inhibitors Arjan W. Griffioen Angiogenesis Laboratory, Department of Pathology, Research Institute for Growth and Development (GROW), Maastricht University and University Hospital, P.O. Box 5800, 6229 HX Maastricht, The Netherlands (E-mail: [email protected]) Using a combination approach employing basic folding principles and incorporating short sequences from the ßsheet domains of anti-angiogenic proteins, novel b-sheet-forming peptide 33 mers have been designed. One of these designed peptides, anginex, is observed to be potently anti-angiogenic. The mechanism of action of anginex was found to be the induction of anoikis leading to apoptosis in angiogenically activated endothelial cells, resulting in an up to 90% inhibition of migration in the wound assay. Anginex inhibited angiogenesis as demonstrated in the in vitro mouse aortic ring assay. In addition, tumor-induced angiogenesis in the chick chorioallantoic membrane was markedly inhibited. Anginex showed profound anti-tumor activity in the syngeneic mouse B16F10 melanoma model and in a xenograft human tumor model. Microvessel density determination as well as magnetic resonance imaging showed that the anti-tumor activity in these tumor models resulted from the anti-angiogenic activity of anginex. A complete absence of toxicity was observed in these models. The data presented here demonstrate that anginex is a promising agent for further clinical development. Based on structure-activity relationships of the angiostatic b-sheet forming peptide anginex, we have designed a (partial) mimetic, 6DBF7, which inhibits angiogenesis and tumor growth in mice. 6DBF7 is composed of a bsheet-inducing dibenzofuran turn-mimetic and two short key amino acid sequences from anginex. This novel anti-angiogenic molecule is more effective in vivo than parent anginex. In a mouse xenograft model for ovarian carcinoma, 6DBF7 is observed to reduce tumor growth by up to 80%. It is suggested that the activity is based on anti-angiogenesis because in vitro tube formation is inhibited and because treatment of mice led to a significant reduction in microvessel density within the tumor. This peptide-mimetic is the first molecule designed as a substitute for an angiogenesis inhibitory protein.

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L39 Concept-based discovery and profiling of PTK787/ZK222584, a novel and orally active angiogenesis inhibitor entering phase III studies K.-H. Thierauch Experimental Oncology, Schering AG, 13342 Berlin, Germany (E-mail: [email protected]) Angiogenesis is considered as a prerequisite for tumor growth. VEGF signaling has been recognized as an indispensable growth factor for angiogenesis qualitatively and quantitatively. Even the heterozygous knock out of the VEGF gene leads to lethal defects during embryonic development. ZK 222584/PTK787 is an efficacious inhibitor of VEGF signaling through its receptors inhibiting angiogenesis and thereby tumor growth. The kinase inhibitor is highly selective for VEGF receptor tyrosine kinases, PDGFR, c-kit and c-fms and is very well tolerated (Wood et al. 2000, Cancer Res. 2000 Apr 15; 60(8): 2178
89). ZK 222584/PTK787 is effective as growth inhibitor in a variety of tumor models of different tissue origin. It was applied in a human prostate tumor model (CWR22) and compared with standard prostate tumor therapy (cyproterone acetate and prostatectomy). ZK222584/PTK787 dose dependently blocks tumor growth (70% inhibition) at an oral dose of 50 mg/kg x day. A co-therapy of antiandrogen (cyproterone acetate) with ZK222584/ PTK787 resulted in increased effectivity. Blood of prostate cancer xenograft bearing mice was collected for analysis of PSA-levels. We found that plasma levels of PSA and tumor size corresponded closely to tumor size reduction (140 ng/ml to below 20 ng/ml). The effect on prostate tumor growth is paralleled by a reduction of the plasma levels of the surrogate marker PSA. The data demonstrate that ZK 222584/PTK787 is an efficacious antiangiogenic antitumor agent, effective via the inhibition of VEGF receptor kinase. ZK 222584/PTK787 given to patients in phase I studies was very well tolerated resulting in few side effects. First signs of efficacy are obtained. Recently two phase III studies were initiated for the treatment of colon cancer in combination the chemotherapeutics Oxaliplatin/Leucovorin/5FU. This work is a result of the joint Schering AG, Berlin, Novartis AG, Basel and KTB Freiburg, Research Project on Angiogenesis-Inhibitors.

L40 Hsp90 transfection reduces ischemia/reperfusion-induces myocardial dysfunction via reciprocal eNOS ser 1177 phosphorylation and Thr 495 dephosphorylation Rabea Hinkel1, Geraldine Daneau2, Philip Raake1, Agnes Brouet2, Peter Boekstegers1, Olivier Feron2 & Christian Kupatt1 1

Medizinische Klinik I, Klinikum Großhadern, Munich, Germany; 2Unit of Pharmacology and Therapeutics, Department of Internal Medicine, UCL Medical School, Brussels, Belgium (E-mail: [email protected]. uni-muenchen.de) The interaction of the heat shock protein Hsp90 with the endothelial NO synthase (eNOS) has been shown in vitro to account for a sustained production of nitric oxide, e.g. the so-called calcium-independent activation of eNOS. In this study, we hypothesized that overexpression of Hsp90 in a pig model of cardiac infarct could help preserving the myocardium from the deleterious effects of ischemia-reperfusion. Percutaneous liposome-based gene transfer was performed by retroinfusion of the anterior interventricular vein 48 h before LAD occlusion (60 min) and reperfusion (24 h). Results: We found that recombinant Hsp90 expression in the ischemic region of the heart led to a 44% reduction in infarct size and prevented the increase in postischemic LVEDP observed in mock-transfected animals. Regional myocardial function, assessed by subendocardial segment shortening in the infarct region,

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was increased in Hsp90-transfected animals both at baseline and following pacing. Importantly, all these effects were completely abrogated by the administration of the NOS inhibitor L-NAME. We also documented that Hsp90 acted as an adaptor for both the kinase Akt and the phosphatase calcineurin, thereby promoting eNOS phosphorylation on serine 1177 and dephosphorylation on threonine 495, respectively. We further validated in cultured endothelial cells that the formation of the Hsp90-eNOS complex was obligatory for this reciprocal regulation. In conclusion, this phosphorylation-driven mode of eNOS regulation establishes Hsp90 as a promising target for enhanced NO formation, which may efficiently reduce myocardial reperfusion injury.

L41 Overexpression of CUZN superoxide dismutase enhances the synthesis of vascular endothelial growth factor Jozef Dulak Department of Cell Biochemistry, Faculty of Biotechnology, Gronostajowa 7, 30-387 Krakow, Poland (E-mail: [email protected]) Abstract not available

L42 Anti-angiogenic and anti-tumor activity of a novel VEGFR-2 tyrosine kinase inhibitor ZD6474 in a metastatic human pancreatic tumor model C.J. Bruns1, G. Ko¨hl2, M. Guba1, M. Yezhelyev1, U. Wiegand2, A. Ryan3, A. Barge3, E. Geissler2 & K.-W. Jauch1 1

Department of Surgery, University of Munich-Großhadern LMU, Munich, Germany; 2Department of Surgery, University of Munich LMU, Germany; 3AstraZeneca, Macclesfield, UK (E-mail: [email protected])

Background: ZD6474 is a novel, orally available inhibitor of vascular endothelial growth factor receptor (VEGFR)2 (KDR/flk-1) tyrosine kinase activity with additional activity against the epidermal growth factor receptor (EGFR)-1 tyrosine kinase (Wedge S. R. et al. Cancer Res., 2000). Consistent, highly significant inhibition of subcutaneous tumor growth in a histologically diverse panel of human tumor models has been achieved with chronic once-daily administration of ZD6474. Material and methods: The aim of this study was to evaluate ZD6474 in a mouse model of metastatic pancreatic cancer. Nude mice (9
10/group) were injected with 1 · 106 L3.6pl human pancreatic cancer cells into the pancreas. Eight days after tumor cell injection, treatment was initiated with vehicle alone, gemcitabine (Gem) (100 mg/kg by i.p. twice weekly), or ZD6474 (50 mg/kg daily by oral administration). Animals were culled 24 days after starting treatment. Results: Compared with controls (1231 mg), tumors reached a mean weight (mg) of 836 (Gem) and 541 (ZD6474). Lymph node (LN) metastases occurred in 10/10 control and 9/9 Gem-treated animals, but only 3/10 animals treated with ZD6474. Liver metastases occurred in 6/10 control, 4/9 Gem-treated and 1/10 ZD6474treated mice. In the proliferating areas at the periphery of the tumor, microvessel density measured by CD31 staining and proliferation measured by Ki67 were significantly reduced by ZD6474. Conclusion: ZD6474 decreased primary pancreatic tumor growth and reduced both LN and liver metastases compared with control or GEM treatments in this model. These results demonstate that ZD6474 is more effective than GEM in this model of human pancreatic cancer.

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The Endothelium in Infectious Diseases Sunday, July 20, 2003, 09.00
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L43 Oxidized phospholipids inhibit immunostimulatory properties of LPS Norbert Leitinger, Stefanie Kirchberger, Stefan Blu¨ml & Johannes Sto¨ckl Department of Vascular Biology and Thrombosis Research; Department of Immunology, University of Vienna, Austria (E-mail: [email protected]) Lipopolysaccharide (LPS), an integral part of the cell wall of gram-negative bacteria, is a potent activator of the innate and adaptive immune system through activation of endothelial cells (EC) and dendritic cells (DC) through Toll-like receptor 4 (TLR-4). EC respond to LPS with upregulation of inflammatory gene expression and DCs gain the capacity to stimulate antigen specific naive T-cells for proliferation. We have shown previously that in EC oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-3phosphorylcholine (PAPC) block LPS-induced activation of inflammatory signaling pathways involving the NFjB as well as p38 MAP kinase pathways. This inhibition led to downregulation of inflammatory adhesion molecules such as E-selectin and VCAM-1, resulting in abrogation of leukocyte adhesion to EC. Furthermore, we could show that oxidized phospholipids inhibit LPS-induced endothelial activation and innate immune response in vivo. Investigating the effects of OxPAPC on adaptive immune responses, we found that oxidized phospholipids inhibit LPS-mediated upregulation of costimulatory molecules and cytokine production in DC. In addition, OxPAPC blunted LPS-induced NFjB and MAP-kinase activation in DC, leading to abrogation of their T-cell stimulatory capacity. Thus, we hypothesize that oxidized phospholipids that are generated during bacterial infections may play an important role in controling massive inflammation and may be important in limiting the maturation of DCs, thereby reducing activation of the adaptive immune system.

L44 Inhibition of Kaposi’s sarcoma (KS) by blocking endothelial cell and KS cell invasion with HIV protease inhibitors Paolo Monini & Barbara Ensoli Laboratory of Virology, Istituto Superiore di Sanita`, viale R. Elena 299, Rome, Italy (E-mail: [email protected]) Kaposi’s sarcoma (KS) is an angioproliferative disease occurring in several clinical-epidemiologic forms including indolent forms, which arise in elderly people of Mediterranean origin and transplanted patients, and aggressive and disseminated forms that develop in young males form subequatorial Africa or in the setting of the HIV infection. All forms of KS are characterized by infiltration of inflammatory cells producing Th1-type cytokines, endothelial cell activation, intense angiogenesis, and presence of spindle shaped cells (KS spindle cells, KSC) that are latently infected by the human herpesvirus 8 (HHV8) and are considered to be the tumor cells of KS. KSC are a heterogeneous cell population dominated by activated vascular and lymphatic endothelial cells mixed with cells of macrophagic origin. Furthermore, some KSC co-express markers of macrophages and endothelial cells such as the VE-cadherin, thus resembling the sinus-lining endothelial-macrophages present in spleen and lymph nodes. Cells expressing this phenotype can also be cultured from PBMC of patients with KS or at risk for KS or, at lower numbers, from normal blood donors. These cells may thus represent circulating KSC progenitors that may be recruited into tissues leading to the typical appearance of multiple lesions at independent sites.

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Evidence indicates that inflammatory cytokines (IC) upregulated both at the systemic level and in tissues of patients with KS or at risk of KS can trigger KS lesion formation. In fact, they activate endothelial cells leading to adhesion and tissue extravasation of lympho-monocytes, induce spindle cell formation and, by inducing angiogenic factors such as bFGF and VEGF, promote angiogenesis. In addition, they reactivate latent HHV8 leading to infection of both circulating and tissue cells. This induces immune responses toward HHV8 that, in risk individuals, are inefficacious against the virus and may paradoxically exacerbate the reactive process and the production of IC. These data, and evidence of spontaneous KS or lesion regression in some patients, indicate that early KS is a reactive process of polyclonal nature. However, in time and in the presence of immunodeficiency, it can progress to a true sarcoma. This is likely due to the long-lasting expression of HHV8 latency genes in spindle cells associated with the deregulated expression of oncogenes and oncosuppressor genes and, for AIDS-KS, with the effects of the HIV-1 Tat protein that, by binding the a5b1 and avb3 integrins, increases the effects of angiogenic factors. A reduced incidence or the regression of KS has been described in HIV-infected patients treated with HIVprotease inhibitors (PI). Our recent studies have shown that systemic administration of PI including indinavir or saquinavir blocks the development and induces regression of angioproliferative KS-like lesions promoted by the inoculation of primary human KS cells or bFGF in nude mice. This is mediated by the inhibition of endothelial cell and KSC invasion and matrix metalloprotease-2 proteolytic activation that occur at the same PI concentrations present in plasma of treated individuals. These data indicate that PI are potent anti-angiogenic molecules that should be exploited also in non-HIV KS and evaluated for blocking angiogenesis and tumors in HIV infected and non-infected individuals.

L45 The cell tropism of human cytomegalovirus (HCMV) depends on the efficiency of a nocodazol-sensitive transport of penetrated virus particles towards the cell nucleus Yolaine Cavignac, Margarete Digel, Kerstin Laib Sampaio & Christian Sinzger Institut fu¨r Medizinische Virologie, Universita¨t Tu¨bingen, City, Germany (E-mail: [email protected]) The infectivity of nonendotheliotropic HCMV/F strains in endothelial cells was found 100
1000 fold reduced when compared to endotheliotropic HCMV/E strains. While no interstrain differences were detected during viral entry, nuclear translocation of penetrated viral DNA in endothelial cells was 100
1000 fold reduced with nonendotheliotropic strains when compared to endotheliotropic strains. Simultaneous staining of viral particles and viral IE-gene expression revealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells are determining the endothelial cell tropism of HCMV. We have therefore evaluated the impact of depolymerization of microtubules (by nocodazole treatment) or actin fibres (by cytochalasin D treatment) on the nuclear translocation of penetrated HCMV/E and HCMV/F in fibroblasts and endothelial cells. Preliminary evidence shows strong inhibition of nuclear translocation of HCMV by nocodazole resulting in low infection efficiency, whereas cytochalasin D had a minor effect on particle movement and infectivity. In support of these functional data, colocalization of immunostained HCMV/E particles with b-tubulin or actin demonstrated that the majority of particles colocalize with microtubules after penetration and move to the microtubulus organization center within 1 h after infection. Beginning from 2 h after infection, virus particles appear at the nucleus, still colocalized with microtubules. In contrast, HCMV/F particles were not translocated towards the MTOC or nucleus. We hypothesize that interactions of viral tegument or capsid proteins with cellular proteins of the dynein / dynactin complex are critical for cell tropism differences.

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L46 Restriction of herpes simplex virus type 1 replication in human brain microvascular endothelial cells: Role of indolamine 2,3-dioxygenase Walter Da¨ubener, Claudia Woithe, Colin R. MacKenzie, Ortwin Adams & Horst Schroten Institute for Medical Microbiology, Heinrich-Heine-Universita¨t, Du¨sseldorf, Germany (E-mail: [email protected]) One of the first steps in the development of bacterial, parasitical or viral encephalitis is the penetration of the blood-brain barrier, which is compromised of microvascular endothelial cells. We have previously examined the capacity of human brain microvascular endothelial cells (HBMEC) to interact with Toxoplasma gondii and with Staphylococcus aureus. We found that IFN-c activated HBMEC are capable of inhibiting the growth of both pathogens. IFN-c mediated indolamine 2,3-dioxygenase (IDO) activity in HBMEC was found to be responsible for the inhibition the growth both pathogens. IDO catalyses the first and rate-limiting step in the kynurenine pathway of tryptophan degradation, which is the conversion of tryptophan to N-formyl-kynurenine. A strong IDO activity leads to a nearly complete depletion of the essential amino-acid tryptophan at the local site of infection which results in a growth arrest of several pathogens. In further experiments we found that IFN-c activated HBMEC are also able to restrict the replication of herpes simplex-virus. In these experiments virus replication was monitored by quantitative real-time PCR as well as by the estimation of the TCID(50). The supplementation of cultures with L-tryptophan nearly completely abrogates the anti-viral effect mediated by IFN-c, suggesting that IDO mediated tryptophan degradation is involved in the anti-viral effect. In addition IDO mRNA was found in IFN-c activated HBMEC by the use of RT-PCR and IDO protein was found in western blot analysis with monoclonal antibodies. Furthermore IDO enzyme activity was measured using photometrically detection of kynurenine in the cell supernatant. Furthermore we found that costimulation with TNF-a enhances both, IDO activity and the anti-viral effect in HBMEC, while TNF-a alone was not able to induce IDO activity. The exact pathway of IDO mediated anti-viral effect remains unclear, however there are some possible explanations. Firstly, proteins necessary for viral replication contain more tryptophan than do the host proteins. Secondly, a reduced amount of tryptophan or an enhanced concentration of tryptophan degradation products might induce a ‘danger signal’ in the cells and influence viral replication.

L47 Changing partners in leukocyte recruitment: New adhesion molecules and their coupling during inflammation Klaus T. Preissner1, Sentot Santoso2, Mathias Herrmann3, Angelika Bierhaus4, Sandip M. Kanse1, Andreas E. May5, Peter P. Nawroth4, Robert W. Colman6 & Triantafyllos Chavakis4 1

Institute for Biochemistry, Medical Faculty, Justus-Liebig University, Giessen; 2Institute for Immunology and Transfusion Medicine, Justus-Liebig-University, Giessen; 3Department of Bacteriology and Hygiene, University of the Saarland, Homburg-Saar; 4Department of Medicine I, University Hospital, Heidelberg; 5Deutsches Herzzentrum, Munich, Germany; 6The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA (E-mail: [email protected]) Leukocyte recruitment to sites of inflammation, infection or vascular injury is a complex event, depending on tightly coordinated adhesion reactions between leukocytes, platelets and the endothelium. The expression profiles of different classes of adhesion receptors, their avidity and affinity states as well as their respective counter-receptors and various anti-adhesive components are major determinants for the required cell
cell interactions. In vitro and in vivo experiments involving knock-out mice indicated a pivotal role for the

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glycolipid-anchored urokinase receptor in organizing the adhesive behaviour of the major b2-integrin, Mac-1, in the acute phase of inflammation. Together with the heamostatic factor kininogen that serves a potent antiinflammatory function, two new counter-receptors for Mac-1 were identified: JAM 3 on platelets and endothelial cells as well as the receptor for advanced glycation endproducts, RAGE, on inflamed endothelium. The cell-type specific expression and activation pattern of these molecules allows the spatio-temporal control of Mac-1 binding function, and respective soluble receptors are potent anti-adhesive molecules. Finally, the secreted ‘extracellular adherence protein’ (Eap) from Staphylococcus aureus tightly binds to ICAM-1 (the major Mac-1 counterreceptor) on endothelial cells and could thereby block leukocyte-endothelium interactions, thus representing a potent bacterial survival strategy as well as a prominent anti-inflammatory priniciple. These multifaceted cellular interactions will help to clarify the molecular mechanisms of leukocyte diapedesis and allow to develop novel therapeutic concepts for the intervention of inflammatory vascular disorders.

L48 Invasion and survival of Staphylococcus aureus in endothelial cells A. Schro¨der1, C. von Eiff2 & M. Aepfelbacher1 1

Max von Pettenkofer Institut, LMU Mu¨nchen, Germany; 2Institut fu¨r Medizinische Mikrobiologie, Universita¨t Mu¨nster, Germany (E-mail: [email protected])

Background: Staphylococcus aureus (S. a.) causes a variety of community acquired and nosocomial infections ranging from soft tissue abscesses to sepsis. Although in tissue examinations S. aureus is mostly found extracellularly, invasion into non-professional phagocytes such as fibroblasts, osteoclasts and endothelial cells has been shown in cell culture studies and has been proposed to underly chronic S. a. infections in vivo. Material and methods: The uptake and survival of different clinical and laboratory strains of S. aureus in endothelial cells (HUVEC) and their intracellular distribution was documented via live cell imaging, fluorescence based inside
outside staining and viability assays. To exclude extracellular growth of bacteria experiments were performed under protection with gentamicin and/or lysostaphin. Results: Using HUVEC transfected with GFP-actin we found that uptake of all tested S. aureus strains was associated with actin accumulation around the bacteria giving rise to so called phagocytic cups. Within 45 min the majority of bacteria became internalised and could be detected in lysosomal compartments. Thereafter, most of the clinical S. a. isolates killed HUVEC within 24 h. Conversely, most non toxic S. a. strains were killed by the endothelial cells within 2
5 d. However, video imaging demonstrated that also under these conditions many endothelial cells became apoptotic and were released from the culture dish. The released HUVEC were immediately replenished by migrating and proliferating cells from the vicinity. Interestingly, one small colony variant survived within HUVEC about 5
10 times better than the other non-toxic strains. Conclusions: Endothelial cells can kill S. a effectively by lysosome associated mechanisms. Upon prolonged infection some S. a. strains kill endothelial cells, likely through unspecific toxicity and/or induction of apoptosis. Some small colony variants can resist killing by mechanisms that need to be further investigated.

L49 Neisseria meningitidis-induced inflammatory cytokine release from human brain endothelial cells involves stress response kinases Olga Sokolova1, Ruth Ja¨gerhuber1, Martin Eigenthaler2 & Alexandra Unkmeir1,3 1

Institute of Hygiene and Microbiology, 2Institute of Clinical Biochemistry, University of Wuerzburg, Germany; Institut fu¨r Mikrobiologie und Hygiene der Universita¨t Wu¨rzburg, Josef-Schneider-Str. 2, 97080 Wu¨rzburg, Germany (E-mail: [email protected])

3

Crucial mechanisms in the pathogenesis of bacterial meningitis are the direct interaction of Neisseria meningitidis with and the consecutive traversal across blood-brain barrier endothelium. Both steps are accompanied by increased production of proinflammatory cytokines, including interleukin 6 (IL-6), IL-8 and

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tumour necrosis factor alpha (TNF-a) production. We have previously shown that invasion of meningococci is mediated by the outer membrane protein Opc that binds fibronectin, thereby anchoring the bacterium to the integrin a5b1 receptor on the endothelial cell surface. However, subsequent signalling pathways involved in bacterial invasion and/or inflammatory endothelial responses still need to be characterised. In the present study, we investigated whether known integrin-regulated signalling pathways in human brain-derived endothelial cell (HBMECs) are regulated by a representative strain of the hypervirulent ST-32 complex meningococci MC58 or its isogenic unencapsulated mutant, termed MC58 siaD. Adherence to and internalisation of N. meningitidis strains MC58 or MC58 siaD into HBMECs activated p38 MAP Kinase and its substrate MAPKAP kinase 2, as well as c-Jun-N-terminal kinases 1 and 2 in a timedependent manner. In contrast, no activation of protein kinase C (PKC) or p42/44 MAP Kinases was detected. Neisseria-induced activation of endothelial p38 MAP Kinase increased mRNA transcription and protein expression of the neutrophil chemoattractant IL-8 and IL-6, since specific p38 MAP Kinase inhibitors SB 203580 and SB 202190 reduced IL-8 and IL-6 release by about 70 and 55%, respectively. Inhibition of p38 MAP Kinases had not effect on bacterial adhesion and invasion. In contrast, the general Protein Tyrosine Kinases (PTK) inhibitor Genistein inhibited both Neisseria invasion and IL-8/IL-6 release, indicating that this group of signalling molecules is involved in both bacterial invasion and cytokine release. Uncovering the molecular mechanisms of N. meningitidis
endothelial interactions is the first step in understanding the pathophysiologic mechanisms that lead to bacterial invasion. This knowledge may provide the basis for the development of new future treatment options of this severe disease.

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Endothelial Progenitor Cells Friday, July 18, 2003, 14.00
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P1 Isolation, characterization and differentiation of endothelial precursor cells from umbilical cord blood Nelli Baal1, Olin D. Liang1, Klaus T. Preissner2, Uwe Lang1 & Marek T Zygmunt1 1

Department of Obstetrics and Gynecology, 2Department of Biochemistry, University of Giessen, Giessen, Germany (E-mail: [email protected]) The placental vascular system develops through two distinct processes: vasculogenesis and angiogenesis. During vasculogenesis, endothelial progenitor cells-angioblasts-from a primitive vascular network. It has been shown that human umbilical cord blood (UCB) contains a significant number of primitive haematopoetic precursor cells (HSC) which may be a reservoir for placental vascular formation. Since HSC from UCB are poorly characterized, we studied proliferation and endothelial differentiation af UCB CD133+ progenitor cells in vitro. UCB was obtained from deliveries. Mononuclear cells were purified using Ficoll gradient centrifugation followed by immunomagnetic cell sorting (MACS). CD133+ UCB cells, a subset of CD 34+ hematopoietic progenitors, were grown on fibronectin-coated, extracellular matrix-coated or mouse fibroblasrt feeder selllayered chamber slides in ECBM-MV2 medium supplemented with fetal bovine serum, horse serum, stem cell growth factor, vascular endothelial cell growth factor, basic fibroblast growth factor and insulin-like growth factor1, in combination with various cytokines and novel angiogenic factor including AFP and hCG (Zygmunt et al., 2002). The phenotype of the cells was determined by flow cytometry using different endothelial and stem cell markers (e.g. CD 34, CD 133, CD 31, CD 141, CD 106, CD 144, vWF). After 3 weeks of culture, the initial cell suspension became adherent, formed a monolayer with a typical EC morphology, and bound fluorescein labeled Ulex europaeus EC-specific agglutinin-1 (VEA-1). We also tested the influence of AFP and hCG an the induction of edothelial phenotype in HSC. Our results suggest that CD133+ progenitor cells from umbilical cord blood can give rise to proliferative ECs, which can be used for a variety of purposes, including steady of vasculo- and angiogenesis during pregnancy.

P2 Erythropoietin regulates endothelial progenitor cells Ferdinand H. Bahlmann, Kirsten deGroot, Jens-Michael Spandau, Aimee L. Landry, Barbara Hertel, Thorsten Duckert, Sascha M. Boehm, Hermann Haller & Danilo Fliser Division of Nephrology, Department of Internal Medicine, Hanover Medical School, Hannover, Carl-NeubergStrasse 1, 30625 Hannover, Germany (E-mail: [email protected]) Background: Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neo-angiogenesis. We tested the hypothesis that erythropoietin (EPO) stimulates proliferation and differentiation of EPCs. Materials and Methods: We assessed circulating CD34+ stem cells (cSC) in whole blood using flow cytometry, and proliferation/differentiation of EPCs in an in vitro assay during 8 weeks of a standard recombinant human EPO (rhEPO) therapy in 11 patients with renal anemia. Results: Treatment with rhEPO induced a significant temporary increase in the number of CD34+ cSCs (week 2: 152%  11%; and week 4: 140%  14%; both P < 0.01 vs. baseline), their number returned to baseline

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levels after 8 weeks. Moreover, rhEPO markedly increased the number of functionally active EPCs already after 2 weeks of therapy (312%  31%; P < 0.01 vs. baseline). This effect was sustained during the whole observation period (week 4: 276%  40%; week 6: 318%  56% ; week 8: 308%  40%; all P < 0.01). The effect of EPO on EPC proliferation and on their functional activity (tube formation assay) is dose dependent. Conclusions: These findings demonstrate for the first time that the cytokine EPO markedly enhances EPC proliferation and differentiation. Currently the pro-angiogenic cytokine EPO is widely used for treatment of cancer anemia without any documented adverse effect on tumor growth. Thus stimulating EPCs using rhEPO could be a safe way to increase the number of functionally active EPCs and may therefore be a novel therapeutic approach in patients with cardiovascular pathology.

P3 The role of B-Raf protein in endothelial cell differentiation Kiril Bidzhekov, Bernd Engelmann & Antonis Hatzopoulos Vaskula¨re Biologie und Ha¨mostase, Institut fu¨r klinische Chemie, LMU-Mu¨nchen, Vascular Genetics Group, GSF Ha¨matologikum, Mu¨nchen, Germany (E-mail: [email protected]) Background: The serine-threonine protein kinase B-Raf plays a key role in the Ras-MAPK signal transduction pathway. It is part of the Raf family, which consists of three proteins A-Raf, B-Raf and Raf-1 (C-Raf). B-Raf is involved in the function of neuronal and endothelial cells. This renders B-Raf an interesting target for studying and regulating the MAP kinase pathway. The aim of the present study was to understand the role of B-Raf in endothelial cells. B-Raf knock out mice die at around E12 and show vascular defects, demonstrating that B-Raf is critical for vascular morphogenesis. Materials, methods and results: Using molecular cloning techniques, we constructed a fusion molecule that contains the kinase domain of B-Raf fused to the estrogen binding site of the estrogen Receptor (ER-B-Raf). By this way the B-Raf activity can be induced by estrogen. The constructs have been sequenced to verify the proper fusion of the relative protein domains. For this purpose we transfected Mouse Embryonic Endothelial Progenitor Cells (eEPCs) by two methods: electroporation, for stable transfections and ‘Lipofectamine 2000’, for transient transfections. The constructs showed good expression levels. Activation by estrogen induced the phosphorylation of MEK1/2, indicating the downstream functional activity of the constructs. Changes in gene expression were analyzed by RT-PCR using a bank of primers that amplify endothelialspecific genes. Our results show that the B-Raf kinase domain is important for the regulation of a set of genes (vWF, Flk-1, Thrombomodulin, Tie-1, M-CSF) responsible for the differentiation of the endothelial progenitor cells. Conclusions and outlook: Modulation of B-Raf activity regulates important genes involved in angiogenesis and therefore B-Raf inhibitors or stimulators might be used to regulate blood vessel growth in the future. In subsequent experiments, we plan to analyze the full spectrum of B-raf regulated genes in endothelial cells by screening cDNA macroarrays.

P4 An in vitro protocol for the differentiation of murine embryonic stem cells into endothelial cells M. Festag,z C. Schleger & B. Viertel Boehringer Ingelheim Pharma GmbH and Co. KG, Department of Non-clinical Drug Safety, 88397 Biberach, Germany (E-mail: [email protected]) Murine embryonic stem (ES) cells are pluripotent cells which are able to differentiate into cells of all three germ layers, namely the endoderm, the ectoderm and the mesoderm. The goal was to develop a protocol for the differentiation of murine ES cells into endothelial cells.

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ES cells were cultivated either in a spinner flask or in hanging drop culture whereby cells aggregated and differentiated into so called embryoid bodies (EBs). The EBs from the hanging drop culture were either plated or transferred into suspension culture. EBs kept in the spinner flask or the suspension culture for longer than 5 days became necrotic and/or apoptotic within their central areas. Since there were no such changes in EBs generated by the hanging drop culture with plating on day 5 of differentiation this method was applied for further experiments. Growth factors, either VEGF alone or VEGF, bFGF, IL-6 and erythropoietin together were tested for their ability to induce differentiation of ES cells into endothelial cells. Different periods of treatment and two different growth factor concentrations were examined. Induction of cell differentiation was investigated by RT-PCR for endothelial cell specific markers comprising VE-Cadherin, PECAM-1, Flk-1, Flt-1, Tie-2 and sFlt over time. In addition, VE-Cadherin and PECAM-1 gene expression levels were quantified by TaqMan PCR. PECAM-1 protein expression level was determined by immunohistochemistry, using laser scanning microscopy. A slight increase in gene expression of these genes was observed in untreated EBs over time. From day 8 of differentiation onwards, a strong increase was seen after treatment with VEGF, compared with the untreated control. Treatment with the growth factor cocktail resulted in slightly higher gene expression levels compared to treatment with VEGF alone. The assay procedure was further optimised by showing that treatment with the highest growth factor concentration from day 5 of differentiation resulted in the same gene expression levels compared with treatment from day 0 onwards. In conclusion, a protocol for the differentiation of ES cells into endothelial cells has been successfully developed. EBs from the hanging drop culture are kept in suspension from day 3 until day 5 and are plated and treated with growth.

P5 ICAM-1 downregulation in tumor endothelial cells is caused by promoter hypermethylation Debby M.E.I. Hellebrekers, Manon van Engeland & Arjan W Griffioen Angiogenesis Laboratory, Department of Pathology, Maastricht University, Maastricht, The Netherlands (E-mail: [email protected]) Background: Tumor endothelial cells(ECs) have a decreased expression of EC adhesion molecules (EAM) involved in leukocyte binding such as ICAM-1, VCAM-1, E-selectin and CD34. This downregulation is explained by exposure of tumor EC to tumor-derived proangiogenic factors such as bFGF and VEGF. Angiogenic factors also severely hamper upregulation of EAM by the proinflammatory cytokines tumor necrosis factor a (TNFa), interleukin-1 and interferon-c. This EC anergy reduces leukocyte adhesion to endothelium and therefore angiogenic factors are involved in tumor escape from the immune response. We previously reported that angiogenic stimulation of EC downregulates ICAM-1 both at the protein and mRNA level. The ICAM-1 promoter contains CG islands, which can be targets for DNA methylation. Therefore, we studied whether epigenetic mechanisms such as DNA methylation are responsible for ICAM-1 silencing by angiogenic factors. Materials and methods: ICAM-1 expression was studied in non-stimulated HUVEC, tumor conditioned HUVEC and tumor conditioned HUVEC treated with the demethylation agent 5-aza-2¢-deoxycytidin (DAC) at 200 nM concentration. Flow cytometric analysis and real time pcr were used to study ICAM-1 protein and mRNA expression, respectively, in these cells. Methylation of the ICAM-1 promoter was studied by bisulfite sequencing of the promoter. Results: DAC induced ICAM-1 expression in tumor conditioned HUVEC at the mRNA level. This also led to upregulation of the ICAM-1 protein, which was assessed by flow cytometry. Bisulfite sequencing showed methylation of the ICAM-1 promoter in tumor conditioned HUVEC, whereas hardly any methylation was observed in non-stimulated HUVEC. Conclusion: Our results show that demethylation increases ICAM-1 transcription in tumor conditioned HUVEC. Together with our bisulfite sequencing results, this indicates that angiogenic factors induce EC anergy by methylation of the ICAM-1 promoter. This may provide a tool for improvement of immunotherapy for cancer. Current studies are investigating the role of this phenomenon in the down regulation of other EAM.

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P6 Isolation, culture and characterization of human endothelial progenitor cells Mihail Hristov, Wolfgang Erl & Peter C. Weber Institut fu¨r Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians Universita¨t, Mu¨nchen, Germany (E-mail: [email protected]) Background: Endothelial progenitor cells (EPC) play a role in vascular repair and may contribute to neovascularization of ischemic tissue. However, data regarding the number of these cells in healthy subjects are scanty and varying. Therefore we have isolated and characterized human EPC from peripheral blood. Material and methods: Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from 150 ml of anticoagulated peripheral blood from healthy male donors, nonsmokers (n ¼ 4). The cells were seeded (2 · 106/cm2) on 24-well plates or chamber slides coated with human fibronectin in endothelial basal microvascular medium with 5% FCS and supplements. After 24 h nonadherent cells were removed and transferred to fibronectin-coated plates. At day 7 in culture, adherent cells were analyzed by fluorescence microscopy and flow cytometry. Results: After 7 days in culture some of the PBMC attached and developed a spindle-shaped appearance. The endothelial-like subpopulation of spindle-shaped attached cells was double positive for DiI-Ac-LDL and lectin (12.3  2.6 cells/field, magn. 20·). We found that EPC are characterized by expression of CD34 (13.1%  2.7%) and vascular endothelial growth factor receptor-2 (18.8%  7.1%). Von Willebrand factor was almost not detectable intracellularly. CD68 staining demonstrated that 22.4%  12.6% of the adherent cells were macrophages. Furthermore, analysis of floating cells after 5 days in culture demonstrated that 45.3%  16.7% express CD4 and they were therefore characterized as lymphocytes. These lymphocytes begin to die during the culture time (11.3%  2.6% Annexin-V/CD4 double positive cells). Conclusions: We isolated human EPC from the PBMC-fraction using adherence culture and endothelial cell specific low serum medium. These factors facilitate the growth of cells with the endothelial phenotype. Collection and reseeding of the nonadherent cells could be of particular interest to specifically increase the efficiency of the isolation procedure. Our data represent additional information regarding the mode of isolation, the number and the characteristics of human EPC. This may help to explain some of the discrepancies reported with respect to the number and characteristics of this subset of cells in the circulation.

P7 Role of embryonal EPCs in chronic hindlimb ischemia of rabbits Achim Pfosser1, Corinna Lebherz1, Mark Thalgott1, Philip Raake1, Matthias Lamparter2, Peter Boekstegers1, Antonis Hatzopoulos2 & Christian Kupatt2 1

Medizinische Klinik I, Klinikum Großhadern; 2Ha¨matologikum GSF Munich, Germany (E-mail: [email protected])

Backround: Induction of therapeutic arteriogenesis and angiogenesis has been reported by application of VEGF or bFGF protein or cDNA. More recent reports indicate that circulating adult endothelial progenitor cells (EPCs) are capable of contributing to capillary growth and improve blood flow in ischemic tissue. Adult EPCs expanded ex vivo and reintroduced into the recipient organism appear as therapeutic tool for otherwise intractable chronic vascular disease. Embryonal EPCs have been suggested as an alternative approach, overcoming shortage of cell supply while displaying immunoprivilege caused by a lack of MHC I expression. Therefore, we used embryonal mice EPCs to study induction of angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model. Methods: In rabbits (n ¼ 5 per group), the external femoral artery was excised at day 0. At day 7, either saline solution or EPCs (5 · 106) or bFGF (50 lg) was infused into the ischemic tissue retrogradely through the anterior

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tibial vein. At day 35, analysis of passage time of contrast agent from the internal iliac artery to the anterior tibial artery was performed in vivo. Angiographic analysis of collateral growth as well as histologic assessment of capillary/muscle fiber ratio were conducted. Results: Embryonic EPCs labeled with Di-I were detected in capillaries of the ischemic limb 5 days after infusion. Moreover, compared to controls, EPC treated hindlimbs displayed an increase in capillary/muscle fiber ratio at day 35 (1.41%  0.09% vs. 0.87%  0.11%). An increased number of collaterals was detected (203%  21% vs. 107%  7% of number counted at day 7), and blood flow velocity increased to 151%  5% of the d7 level in the EPC-treated group, whereas levels did not increase in the control group (109%  10%). bFGF application resulted in similar increase in capillary density (1.56%  0.19%), collateral growth (161%  26%) and blood flow velocity (143%  13%). Conclusion: We conclude that mice embryonal EPCs are capable of inducing angiogenesis and arteriogenesis in a rabbit model of chronic ischemia, similar to the effect observed with bFGF. Wether embryonal EPCs mainly release growth factors and thereby induce angiogenesis and arteriogenesis or utilize another angiogenic signaling pathway remains to be determined.

P8 Isolation and characterization of porcine yolk-sac-derived endothelial cells Johanna Plendl1 & Robert Auerbach2 1

Freie Universita¨t Berlin, Fachbereich Veterina¨rmedizin, Institut fu¨r Veterina¨r-Anatomie, Berlin, Germany Laboratory of Developmental Biology, University of Wisconsin, Madison, Wisconsin, USA (E-mail: [email protected])

2

Background: The yolk sac is the first site of vasculogenesis. The aim of our study was to establish cell lines that could help us understand the nature of endothelial differentiation and that might have potential use in xenotransplantation. Material and methods: Cells were isolated from porcine yolk sacs of day 18 embryos. Initial cultures consisted of adherent and non-adherent cells. Endothelial cell lines were established from adherent cells and examined by specific antibody and lectin labelling. Cells were pre-labeled with CM-dil cell tracker, mixed with S180 sarcoma cells and inoculated into irradiated mice. UV illumination was used to identify yolk sac-derived endothelial cells. To determine whether MHC antigens could be upregulated by cytokines, cells were incubated in recombinant human interferon-gamma. Results and conclusions: Three-dimensional tube formation was observed in cells and Weibel Palade bodies as well as cellular receptors for acLDL and angiotensin converting enzyme were found. Primitive endothelial cells from the porcine yolk sac express MHC I but not class II MHC antigens, considered the most potent stimulators of immune-mediated graft rejection, they retain the ability to form blood vessels on transplantation into immunocompromised host animals, do not express and cannot be activated to express VCAM, involved in graft rejection. However, these cells express integrins involved in immune-mediated graft rejection and bind Bandeiraea simplicifolia lectin, a target of hyperacute rejection.

P9 Vascular remodeling is linked to an increased load of the endoplasmic reticulum Felicitas Pro¨ls, Bernhard J. Berger, Tina Mu¨ller, Ivo R. Buschmann, Kirsten Peters, Matthias Kirsch & Bodo Christ Institute of Anatomy and II, Albert Ludwigs-University, D-79104 Freiburg, Germany (E-mail: felicitas.proels@ anat.uni-freiburg.de) There is increasing evidence that molecular chaperones are specifically involved in the control of vascular growth and remodeling. The molecular chaperones that reside within the endoplasmic reticulum (ER) are of specific

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interest since all secreted proteins (e.g. growth factors, extracellular matrix proteins) have to pass the quality control system in the ER. Increased ER load, as it occurs during vascular remodeling, results in enlargment of the ER and an increased synthesis of specific chaperones. Recently a novel chaperone of the Hsp40 family, named Mdg1, has been identified by us. Mdg1 is highly conserved in all vertebrates tested. Here, we show that Mdg1 is generally expressed at low levels in primary cell species, as in human endothelial cells, fibroblasts, skeletal muscle, smooth muscle cells or the HeLa cell line. NO-radicals, as they are released by activated macrophages, CoCl2-application, heat shock, or vascular remodeling as evaluated by in vitro angiogenesis and arteriogenesis go along with increased Mdg1 mRNA levels. While shear stress has no effect on Mdg1 expression levels, stimuli that largely increase the ER load, such as thapsigargin, monensin/ionomycin, tunicamycin, and dithiothreitol strongly induce Mdg1. These increases are most pronounced (20
47 fold) in endothelial cells. Comparison of the temporal profiles of Mdg1 expression with those of the ER-resident Hsp70, BiP, reveal an almost identical kinetic for Mdg1 and BiP indicating that Mdg1 upregulation is closely linked to an increased ER load and thus possible present a novel target for inducing or inhibiting angio- or arteriogenic processes.

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The Endothelium in Development and Angiogenesis I Friday, July 18, 2003, 17.00
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The Endothelium in Development and Angiogenesis II Saturday, July 19, 2003, 08.30
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P10 Functional analyses of the av integrin gene using ‘lacZ knock-in’- and ‘conditional gene knock-out’ av integrin alleles in mice M. Keller1, M. Po¨ckl1, U. Kuhn1, M. Ibsch2, H. Wolburg2 & B.L. Bader1 1

Max-Planck-Institute for Biochemistry, Dept of Molecular Biology, Martinsried, Germany; Tu¨bingen, Institute of Pathology, Germany (E-mail: [email protected])

2

University of

Background: Integrins are a family of cell surface receptors which mediate cell adhesion, coordinate both the cytoskeletal organization and signaling processes. The av integrin subunit can dimerizes with five b subunits b1, b3, b5, b6, and b8. av integrin gene expression and functional analysis using (i) av integrin-specific antibodies and (ii) antagonistic RGD-based peptidomimetics have implicated av integrins in various morphogenetic events during embryonic development and in adult physiology and pathology such as vasculogenesis and angiogenesis as well as tumor angiogenesis. A targeted null-mutation in the av integrin gene in mice is 100% lethal and leads to two classes of vascular phenotypes. (i) Class I (early phenotype): deficient placentation and embryonic lethality at E10.5, (ii) Class II (late phenotype): defective embryonic intracerebral vasculature and brain hemorrhage at E12.5, cleft palate, intestinal haemorrhage at birth and finally perinatal lethality (Bader et al., Cell 1998, 95: 507
19). Since av integrins can be expressed on both endothelial cells as well as on perivascular cells it is not evident which cell type(s) and underlying mechanisms contribute to the vascular defects in av integrin-deficient mice. Materials and methods: To answer these questions we have generated strains of mice carrying a ‘lacZ-knock in’ av integrin allele and a ‘conditional gene knock-out’ av integrin allele in mice. Using these mutant mouse strains, we assessed the av integrin gene expression and cell type-specifically ablated av integrins dependent on nestin-cre and tie1-cre expression in compound transgenic mice. Results and conclusion: We detected aberrant vascular sprouting and the formation of glomeruloid vascular structures in mutant brains and retinae. These findings underline that av integrins are important mediators of vascular morphogenesis during brain and retina development.

P11 Vascular endothelial growth factor-C is secreted from cells in culture and detectable in tumour lysates
a new assay for quantification by sandwich elisa Hiroko Bando1, Maren Brokelmann1, Bence Sipos2, Jonathan Sleeman3 & Herbert A. Weich1 1

Department RDIF, National Research Centre for Biotechnology (GBF), 38124 Braunschweig, Germany; Department Pathology, University of Kiel, 24105 Kiel, Germany; 3Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, 76021 Karlsruhe, Germany (E-mail: [email protected])

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Background: In opposite to vascular endothelial growth factor (VEGF-A), VEGF-C and VEGF-D are specific regulatory factors for lymphatic endothelial development, proliferation and lymphangiogenesis in vivo. Both growth factor mediate their biological activity mainly by the receptor FLT-4/VEGFR-3. However, some proteolytic processed forms of VEGF-C can also bind to KDR/VEGFR-2. VEGF-C and VEGF-D are produced as precursor molecules and finally, N-terminal and C-terminal processed forms are secreted from cells. Material and methods: In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins, recombinant VEGF-C and VEGF-D together with polyclonal antibodies for VEGF-C and VEGF-D. The ligands, soluble receptors and different type of peptide or native protein specific antibodies have been used to develop a microtiter plate-based sandwich assay. Here we describe at the first time a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of human and murine VEGFC. The ELISA can detect human and rat VEGF-C with a minimum detection l imit of 100 pg/ml. The assay does not show any cross-reactivity with the related proteins VEGF-A and VEGF-D. Furthermore, complex formation with its soluble receptor FLT-4 does not diminish the ability to be detected by the antibodies. Results: Using this assay, VEGF-C was measured in the supernatants and lysates of different cell types and in tumour tissues samples of murine and human origin. Normal cell lines in culture secrete VEGF-C in very low amounts (<1 ng/ml) whereas transfected cells can secrete up to 50 ng/ml VEGF-C into the supernatant. In human tumour tissue samples VEGF-C can be detect in some carcinoma lysates the low ng/mg protein range. Conclusions: This ELISA could be a useful tool for investigations concerning the production and physiological function of VEGF-C in lymphangiogenesis and tumour-induced lymphangiogenesis.

P12 Analysis of the hemorrhages caused by Syk-deficiency during embryogenesis and in bone marrow chimeras Barbara C. Bo¨ck, Hartwig Wolburg & Friedemann Kiefer Max-Planck-Institute for Vascular Biology, Mu¨nster, Germany; Institute of Pathology, University of Tu¨bingen, Tu¨bingen, Germany (E-mail: [email protected]) The non-receptor tyrosine kinase Syk plays an important role in immunoreceptor and integrin signaling in hematopoietic cells. Homozygous Syk-deficient embryos develop severe lethal hemorrhages at midgestation (E14.5 and E17.5). Radiation chimeras which are reconstituted with fetal liver derived from Syk-null embryos develop progressive abdominal and ultimately lethal bleedings 3
6 month post reconstitution. In both settings the lymphatic vasculature appears to be affected. While the role of Syk in immunoreceptor signaling is well characterized the mechanisms leading to hemorrhaging have not been deciphered. By immunohistological analysis we were able to show dramatic changes in vessel morphology of Syk-deficient embryos. Furthermore an ultrastructural analysis revealed a thinning of the endothelial lining and abundant necrosis in Syk-null embryos. We have investigated possible endothelial cell-autonomous as well as cell non-autonomous mechanisms as the cause of the vascular defects caused by Syk-deficiency. We used polyomavirus middle T-antigen transformed endothelial cells (endothelioma cells), which express low levels of endogenous Syk, as a model for immature endothelium. To analyze a possible signaling function of Syk in endothelial cells we derived Sykdeficient endothelioma cell lines as well as several variants thereof which were re-transfected with mutant versions of Syk. We found a modulation of Syk kinase activity in endothelioma cells depending on the extracellular matrix on which these cells were grown. Furthermore, RT-PCR analysis suggested that depending on the presence or absence of Syk expression of endothelial markers can be modulated. To probe for a possible non-endothelial cell autonomous function of Syk in the pathogenesis of hemorrhages, we are analyzing the expression of growth factors by cells of the hematopoietic compartment. Data on this analysis will be presented. We propose a mechanistic involvement of both endothelial cell autonomous and cell non-autonomous mechanisms in the bleeding pathogenesis of Syk-deficient embryos and bone marrow chimeras. Impaired endothelial function could involve an altered response to the extracellular environment and consequently an altered gene expression profile and morphogenetic behavior. Cell non-autonomous functions could involve altered survival and inappropriate differentiation of immature endothelial cells.

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P13 Isolation and collection of single (living) cells by LMPC (laser microdissection and pressure catapulting) Renate Burgemeister, Gabriele Friedemann, Susanne Ehnle, Monika Stich, Ulrich Sauer, Rainer Gangnus & Karin Schu¨tze PALM Microlaser Technologies, Am Neuland 9 + 12, 82347 Bernried, Germany (E-mail: [email protected]) Many fields in the area of biology and medicine need the use of very clean samples as a prerequisite for reliable results. Whether in tumour biology, cancer genetics, preimplantation- or prefertilization diagnosis, prenatal diagnosis, and so on, pure samples are indispensable. It is now approved that whole tissue specimens have important disadvantages when they are used e.g. for gene expression analyses. Using tissue homogenates and bulk material bears the risk of obscuring genetic deviations and therefore expression changes of an individual cell type by the mass of surrounding cells. In particular, microarray analyses will give excellent results only by use of collected specific samples without any contamination. The LMPC technology (laser microdissection and pressure catapulting) allows microdissection of single cells, cell clusters, chromosomes etc. and their transfer directly into or onto a collection device. These collected cells or chromosomes can be investigated as singles or pooled for molecular or proteomic analyses. Any kind of tissue from various sources (also archival histological samples) and even subcellular structures can be captured using this laser method. Even living cells can be collected in this way and processed further. This new approach can be used to establish a homogeneous cell population out of a heterogeneous cell population (e.g. after transfection experiments). A special software program (Metafer P from MetaSystems) combined with LMPC allows the automated detection and collection of rare events on the object slide (e.g. cells or metaphases) and saves a lot of time in finding e.g. FISH labelled cells.

P14 The NADPH oxidase subunit p22phox is regulated by a redox sensitive pathway in endothelial cells T. Djordjevic1, A. Pogrebniak1, C. Za¨hringer1, R.S. BelAiba1, S. Bonello1, T. Kietzmann2, H. Acker3, J. Hess1 & A. Go¨rlach2 1

Experimentelle Kinderkardiologie, Deutsches Herzzentrum Mu¨nchen an der Technischen Universita¨t Mu¨nchen; Institut fu¨r Biochemie und Molekulare Zellbiologie, Universita¨t Go¨ttingen; 3Max Planck Institut fu¨r Molekulare Physiologie Dortmund, Germany (E-mail: [email protected])

2

Reactive oxygen species (ROS) play an important role as signaling molecules in the vasculature and sustained generation of ROS has been implicated to be involved in pathogenesis of diseases such as atherosclerosis, vascular remodeling including angiogenesis and cancer. NADPH oxidases have been reported to contribute to ROS production in the endothelium, although the underlying mechanisms are not completely resolved. Since ROS generation can occur rapidly within minutes but also delayed within hours of stimulation, we investigated whether ROS play a role in the regulation of the NADPH oxidase in endothelial cells. Two photon confocal microscopy showed that ROS generation was closely related to the expression of the NADPH oxidase subunit p22phox coupled to a fluorescent protein, and ROS production was enhanced by coexpression of the subunit gp91phox. Thrombin stimulation resulted in a rapid increase in ROS generation within 15 minutes, but also in a delayed elevation of ROS production which was maximal after three hours of

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stimulation and which was accompanied by an upregulation of p22phox protein levels. Antioxidant treatment abrogated these responses. Furthermore, H2O2 elevated p22phox protein levels in a time- and dose-dependent manner. Expression of superoxide dismutase (SOD) enhanced ROS production and p22phox protein levels. These responses were inhibited by depletion of SOD. Furthermore, thrombin and H2O2 stimulated proliferative responses in endothelial cells. These data show that p22phox is regulated in a redox-sensitive manner in endothelial cells. ROS generated by rapid activation of NADPH oxidase may further lead to induction of this enzyme resulting in the prolonged generation of ROS. Such a positive feed back mechanism may explain the sustained elevation of ROS observed in atherosclerosis, vascular remodeling and cancer and may thus play a central role in the pathogenesis of these diseases.

P15 Dose dependent effect of TNF-alpha analogue LK-805 on capilary growth and cytotoxicity I. Fonda1, S. Caserman2, V. Gaberc Porekar1, M. Kenig3, T. Lah2 & V. Menart1,3 1

National Institute of Chemistry, Ljubljana, Slovenia; 2National Institute of Biology, Ljubljana, Slovenia; 3Lek d.d., Recombinant Biotechnology Department, Ljubljana, Slovenia (E-mail: [email protected])

Background: Recently we have designed several new TNF-alpha analogues showing increased in vitro cytotoxicity on mouse L-929 cell line. Analogue LK-805, which has a simple mutation E107K in the exposed tip region of the molecule, showed significantly lowered systemic toxicity and increased anti-tumour activity in Sa-1 tumour-bearing mouse models treated with this analogue. A mutation introduced charge reversal and clustering of three lysines in the exposed tip region of LK-805. We assumed that additional ionic interactions between the positively charged TNF analogue and negatively charged components of the cell surface were created, which might contribute to improved properties of LK-805. Material and methods: Specific activity of the native TNF-alpha and analogue LK-805 on L929 cells and on heparinase-treated L929 cells was determined. The influence of both molecules on capillary formation and growth were compared on aorta ring models in the presence and absence of VEGF. Results: Analogue LK-805 exhibited approximately 2-fold higher cytotoxicity on L929 cells in comparison to unmodified TNF. Treatment of L929 cells with heparinase prior to the addition of LK-805 led to cytotoxicity comparable to native TNF. When heparan sulfate was added to the medium, enabling competitive binding to LK805, some reduction of cytotoxicity was observed. In the aorta ring assay with analogue LK-805 a significant dose dependent reduction of the cytotoxic activity on newly forming capillaries was observed in comparison to native TNF, in the presence and absence of VEGF. Conclusion: These results indicate that actually some interactions between the oppositely charged LK-805 and cell associated heparan sulfate exist. This can lead to the increased local concentration of the analogue and perhaps also to its prolonged half-life. The reduced cytotoxicity on endothelial cells of newly forming capillaries could have a possible role in the substantially lower systemic toxicity in comparison to native TNF.

P16 Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells Tim Fuller, Thomas Korff, Adrienne Kilian, Gudrun Dandekar & Hellmut G. Augustin Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, D-79106 Freiburg, Germany (E-mail: [email protected]) Contact dependent interactions between endothelial cells (EC) as well as between EC and mural cells play a key role in the formation of a regular vascular system and the assembly of the vessel wall. Recent studies have

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identified ephrinB2 and EphB4 as markers and makers of arterio-venous differentiation during vascular development. Based on these findings, we hypothesized that Eph-ephrin interactions in the vascular system mediate distinct propulsive and repulsive effector functions which provide guidance signals for the proper spatial organization of the developing vasculature. Utilizing a set of specialized endothelial differentiation and angiogenesis assays, the present study was aimed at studying vascular morphogenic functions of endothelial EphB4 and ephrinB2 activation. EphrinB2-Fc acts antiadhesive and induces detachment of EC as evidenced by (1) inhibition of adhesion to ephrinB2-Fc coated culture dishes, (2) detachment of EC grown as differentiated 3D spheroids, and (3) endothelial denudation of explanted fragments of umbilical vein. Conversely, soluble ephrinB2-Fc inhibits lateral cell migration, VEGF gradient-driven chemotaxis, capillary-like network formation and sprouting angiogenesis. In turn, soluble EphB4-Fc is proadhesive and stimulates EC migration and sprouting angiogenesis. EphrinB2 mediated repulsive signals are transduced by EphB4 as demonstrated by EphrinB2-Fc inhibition of sprouting angiogenesis of constitutively EphB4 overexpressing EC. Confrontation experiments of EphB4 overexpressing EC with EC overexpressing full length or truncated ephrinB2 that lacks the cytoplasmic catalytic domain demonstrated that forward EphB4 signaling with EphB4 tyrosine phosphorylation restricts intermingling of cells and supports cellular segregation. Taken together, the data identify distinct propulsive and repulsive effector functions of endothelial ephrinB2 and EphB4 that mediate spatial positional signals during angiogenesis and vessel assembly.

P17 Transcriptional regulation of the endothelial cell specific cytokine angiopoietin-2 Anja Hegen, Stefanie Koidl, Dieter Marme´, Hellmut G. Augustin & Ulrike Fiedler Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center Freiburg, Germany (E-mail: [email protected]) Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are ligands of the endothelial cell receptor tyrosine kinase Tie2. Tie2 activation promotes vessel assembly and maturation, mediates endothelial cell survival and is involved in the recruitment of mural cells. Ang-1 acts in a paracrine manner on endothelial cells by inducing receptor phosphorylation and subsequent signal transduction. In contrast, Ang-2 appears to act preferentially as an antagonist for Ang-1 dependent Tie2 functions. Moreover, Ang-2 is primarily produced by endothelial cells and, thus, acts as an autocrine growth factor. In order to better understand the molecular basis of Ang-2 mediated autocrine activities on endothelial cells, we have performed a structural and functional analysis of the Ang-2 promoter. The human Ang-2 promoter (bp )4427 to bp +476) was cloned and luciferase reporter geneexpression of different length Ang-2 promoter constructs was studied in endothelial cells and nonendothelial cells. These experiments showed that the Ang-2 promoter is capable to drive luciferase reporter expression in endothelial cells but not in nonendothelial cells. Promoter deletion analyses revealed that bp )109 to bp +476 of the Ang-2 promoter are sufficient to mediate endothelial cell specific reporter gene expression. Consistent with Ang-2 mRNA expression data, the reporter gene expression of the short Ang-2 promoter constructs could be stimulated by VEGF, FGF-2, and PMA. Analysis of different length longer promoter fragments identified a potent negative regulatory element located between bp )4427 to bp )2004 of the promoter. This negative regulatory region is also capable to repress the SV40 promoter in both endothelial and nonendothelial cells confirming the identification of a strong negative regulatory element in the Ang-2 promoter. Members of the Ets family of transcription factors were identified as mediators of endothelial cell specific regulation of the Ang-2 promoter: Ets-1 and Elf-1 are able to induce promoter activation when cotransfected with luciferase reporter constructs. Taken together, the experiments reveal unique regulatory properties of the Ang-2 promoter, characterized by (i) endothelial cell specificity-mediating Ang-2 promoter elements, (ii) an involvement of Ets family transcription factors in the regulation of Ang-2 promoter activity and (iii) responsiveness of Ang-2 gene expression to VEGF, FGF-2, and PMA stimulation.

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P18 Differential effect of no and pkc on Cx37-dependent gap junctional communication P. Kameritsch, N. Khandoga & U. Pohl Department of Physiology, Ludwig-Maximilians-University, Munich, Germany (E-mail: Petra.Kameritsch@lrz. uni-muenchen.de) In blood vessels neighboring cells are coupled via intercellular channels (gap junctions, GJ) consisting of connexins (Cx), which play a major role for the regulation of the vascular tone and a coordinated vasoactivity. In the vascular system Cx37, Cx40, Cx43, and Cx45 are expressed and their regulation and specific role for the control of the GJ-permeability is still unclear. We have already shown that NO reduces the GJ-permeability via a partial cGMP-independent, selective effect on Cx37. As NO can modulate directly or indirectly the PKC-activity, we analyzed now whether the NO effect can be mediated via PKC. HeLa-cells, which do not express Cx in their wild type form, were transfected with Cx37 or Cx43 (main Cx’s in the endothelial cells). A GJ-permeable fluorescent dye (Alexa Fluor 488) was injected in a single cell and the dye spreading into neighboring cells was taken as a measure for the GJ-permeability. NO (2 lM SNAP, 30 min) reduced in cocultures of Cx37- and Cx43cells (n ¼ 35) selectively the dye spreading via Cx37 from 41%  3% (con) to 19%  4% (NO) but had no effect in cells coupled via Cx43 only. An activation of PKC using PMA (1 lM, 30 min) reduced the GJ-permeability of Cx37-containing GJ to a similar extent (con: 34%  5%; PMA: 13%  4%). Moreover, in contrast to NO it also blocked the coupling via Cx43. Immunhistochemistry revealed no redistribution of Cx37 away from the cell membrane after NO-incubation. In contrast, after activation of PKC, nearly no Cx37 could be found in the cell membrane after 20 min. These results indicate that the inhibition of the cell
cell-coupling by NO is due to a reduction of the GJ permeability whereas the activation of the PKC is followed by a reduction of Cx37 in the cell membrane. The cGMP-independent effect of NO on the intercellular coupling is apparently not mediated via an activation of PKC.

P19 In vitro stimulation of angiogenesis in isolated pancreatic islets in a threedimensional fibrin matrix A. Kidszun1, D. Erb1, G. Hertl1, G. Breier2, E. Morgenstern3, K. Preissner4, R.G. Bretzel1 & T. Linn1 1

III. Medical Department and Clinic University Hospital Giessen, Germany; 2Max-Planck-Institute for Physiological and Clinical Research Bad Nauheim, Germany; 3University of Saarland, Homburg/Saar, Germany, 4 Biochemical Department University of Giessen, Germany (E-mail: Andre.Kidszun@innere. med.uni-giessen.de) Background: The formation of a new microvasculature to ensure sufficient nutrition and oxygen supply after islet cell transplantation is essential for the long-term survival and function of the graft. During isolation procedure islets are disrupted from their surrounding blood vessels, but maintain single endothelial cells. In this study we examined the angiogenic capacity of isolated pancreatic islets by exogenous stimulation, different culture conditions and genetic modification. Materials and methods: Isolated islets of Langerhans from FVB-mice were embedded in a three-dimensional fibrin matrix, soaked with low serum medium and incubated for 48 h. We investigated the effects of normoxic (5% CO2, 95% humidified air) vs. hypoxic (2% O2, 5%CO2, 93%N2) conditions, VEGF (5 ng/ml) and bFGF (5 ng/ ml) + TNFa (20 ng/ml). Furthermore we surveyed islets from transgenic RIP (rat insulin promoter)
VEGF mice in a high glucose (300 mg/dl) medium. To quantify angiogenesis we defined the islet sprouting index, ISI¼ R length of endothelial sprouts/R living islets. Measurements were made using Olympus
digital photography and Analysis
Software. Statistical significance was proved by means of Mann
Whitney-Test. For histology gels were embedded in paraffin.

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Results: Isolated pancreatic islets built up new endothelial capillary-like structures. Endothelial cells were detected in immunohistochemical lectin stains (Bandeira simplicifolia) and electron microscopy preparations. VEGF and transgenic RIP-VEGF islets showed a 1.7 and 3.3 fold increase of ISI compared to control (P < 0.03, P < 0.02). Hypoxic culture conditions (24 h) and bFGF + TNFa remarkably stimulated angiogenesis and led to a notable 4.8 and 5.4 fold increase of capillary sprouting (P < 0.01, P < 0.001). Conclusion: We show that isolated pancreatic islets are capable of forming new capillary structures and prove their susceptibility to angiogenic stimulation.

P20 Induction of apoptosis in proliferating, but not in confluent human endothelial cells by n-3 fatty acids EPA and DHA Hyo Jung Kim, Claudia Vosseler, Peter C. Weber & Wolfgang Erl Institut fu¨r Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany (E-mail: [email protected]) Background: In a study of cancer cell growth in nude mice, docosahexaenoic acid (DHA) showed potent antiangiogenic properties (Rose and Connolly, Int. J. Oncol. 1999; 15: 1011). It was speculated that this effect could be mediated by induction of apoptosis in endothelial cells (EC). Because it is not known, whether different polyunsaturated fatty acids (PUFA) induce cell death or apoptosis in EC, we investigated the influence of PUFA on the growth of human umbilical vein EC (HUVEC) and vascular smooth muscle cells (VSMC). Materials and methods: HUVEC or VSMC were incubated with arachidonic acid, linoleic acid, eicosapentaenoic acid (EPA) or DHA at different concentrations for 24 h. Cell numbers and viability were measured by trypan blue counting. Apoptosis was detected by binding of Annexin-V and sub-G1 cell cycle analysis by flow cytometry. Results: Treatment of confluent HUVEC with the different PUFA did not show significant effects on cell number or viability. In contrast, incubation of sub-confluent (75%), i.e. proliferating HUVEC or VSMC with DHA increased the number of dead cells. This cell death was due to apoptosis, as demonstrated by an enhanced binding of Annexin-V and an increase in the percentage of cells in the sub-G1 phase of the cell cycle. Among the PUFA tested, n-3 PUFA, especially DHA, were more active as compared to n-6 PUFA. Conclusion: These results suggest that the previously observed anti-angiogenic effect of DHA in a tumor model is most probably due to induction of apoptosis in proliferating EC. These effects are not observed in confluent, resting HUVEC. Further studies are needed to examine the signaling pathway through which n-3 PUFA mediate apoptosis in sub-confluent EC and VSMC.

P21 Shear stress induced release of bFGF from endothelial cells is mediated by proteolytic activities C. Klarskov, T. Gloe, S. Zahler, F. Kro¨tz, H.Y. Sohn & U. Pohl Institute of Physiology, LMU, Munich, Germany (E-mail: [email protected]) Background: Basic fibroblast growth factor (bFGF) is a cytokine stored in large amounts within endothelial cells and plays a pivotal role in vascular remodelling by regulating migration, proliferation, and new vessel formation. Recently, we could show that shear stress induced release of bFGF is mediated by the integrin aVb3 (Gloe et al. JBC 2002). Based on these findings we investigated whether matrix modulation by elastase contribute to signalling cascades leading to enhanced bFGF liberation from endothelial cells. Material and methods: Porcine aortic endothelial cells were kept either under static conditions or subjected to shear stress of 20 dyn/cm2. Conditioned media from static controls or shear stress were tested for elastase activity using the specific substrate peptide Suc-Ala-Ala-Pro-Val-pNA and bFGF using ELISA. Translocation of bFGF from the cytosol to the membrane was quantified with facs measurements. In part of the experiments,

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inhibitory peptides (GRGDSP) of cell matrix interactions as well as the protease inhibitor aprotinin were added. In order to identify members of the underlying signalling cascade kinase inhibitors wortmannin and chelerytrine were tested. Results: Elastase activity was enhanced from 5
15 mU/ml (P < 0.05, n ¼ 6) during 6 h shear stress and bFGF release was 10-fold higher than in static controls (P < 0.05, n ¼ 6). LDH concentration was similar in both, shear stress and static indicating a lack of unspecific membrane damage. Biological activity of released bFGF was verified in bioassays, where only shear stress conditioned medium induced proliferation (P < 0.05, n ¼ 6) and capillary like structure formation (P < 0.05, n ¼ 6). Release of bFGF was significantly reduced by incubation with aprotinin. Moreover, after 2 h of incubating static cultured cells with elastase (200 mU/ml) alone, bFGF release increased to a similar amount as found under shear stress (n ¼ 4). Western blots showed that bFGF indeed was released from EC and not proteolytically from the matrix. Upon exposure to elastase, membrane bound bFGF was increased more than 2-fold. This translocation from cytosol to the membrane could be prevented by inhibition of the integrin aVb3, by wortmannin (PI3 kinase) and chelerytrine (PKC). Conclusion: The results indicate that shear stress is a potent stimulus for release of fully active bFGF that may act as mediator of vascular remodelling. The mechano-sensing and/or release mechanism is critically dependent on matrix modulation via elastase and subsequently by specific cell matrix interactions via avb3 integrins.

P22 Tumorigenic conversion of endothelial cells V. Krump-Konvalinkova1, F. Bittinger2, U. Friedrich3, E. Kleideiter3, S. Yasuda4, G. Tigyi4, C.J. Kirkpatrick2 & W. Siess1 1

Institute for Prevention of Cardiovascular Diseases, Ludwig Maximilian University Mu¨nchen; 2Institute of Pathology, Johannes Gutenberg University Mainz; 3Dr Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart; 4Department of Physiology, University of Tennessee Health Sciences Center, Memphis, Tennessee, USA (E-mail: [email protected])

Background: Tumors of endothelial origin, angiosarcoma (AS), develop very infrequently. Until now, only two ASderived cell line presenting endothelial characteristics could be isolated, ISO-HAS [Masuzawa et al., Int J Cancer 1999; 81: 305
8] and AS-M (unpublished). Here we report on characteristics of AS-derived cell lines that differed from those of freshly isolated endothelial cells. Material and methods: The AS-derived cells lines were characterized by immunofluorescence and RT-PCR. In functional assays, the uptake of DiI-Ac-LDL and angiogenic reaction on matrigel as well as the response to proinflammatory stimuli were assayed and compared to freshly isolated human umbilical vein endothelial cells (HUVEC). Telomerase activity and telomere length were evaluated by telomere repeat amplification protocol (TRAP) and telomere repeat fragment (TRF) length analysis Southern blots respectively. Results: The AS-derived endothelial cell lines presented many of characteristics of freshly isolated HUVEC, such as the expression of von Willebrand factor, CD31, CD34, KDR and Flt-1. However, both AS-derived cell lines differed from HUVEC in a nuclear accumulation of p. 53, in a delayed angiogenic reaction and in a reduced expression of caveolin. The two AS-derived cell lines presented a different expression pattern of lysophosphatidic acid receptors and cell adhesion molecules, cytokines and chemokines responsive to proinflammatory stimuli, possibly reflecting differences in the degree of tumorigenic conversion in these cell lines. Even though the telomeres of both established cell lines were longer than telomeres of freshly isolated HUVEC, neither transcripts encoding telomerase nor telomerase activity were detected. Conclusions: (1) The AS-derived cell lines can substitute for freshly isolated endothelial cells to study some endothelial cell functions in vitro. (2) Tumor cells of endothelial origin use telomerase-independent mechanism for maintaining the telomere length. This observation may have implications for development of therapeutic approaches for angiosarcoma.

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P23 Onco-developmental alpha-fetoprotein (AFP) as a pro-angiogenic factor in the fetomaternal unit Olin D. Liang1,2, Thomas Korff3, Jasmin Rifaat1, Anja Hoffmann1, Friederike Herr1, Klaus T. Preissner2 & Marek Zygmunt1 1

Department of Obsterics and Gynecology, Justus-Liebig-University, Giessen, Germany; 2Department of Biochemistry, Justus-Liebig-University, Giessen, Germany; 3Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center at the University of Freiburg, Freiburg, Germany (E-mail: olin.d.liang@ biochemie.med.uni-giessen.de)

Background: A fully developed vasculature and adequate blood supply in the placenta and uterus is essential for the establishment of a successful pregnancy where angiogenesis and vascular remodeling are crucial processes. The onco-developmental alpha-fetoprotein (AFP) is mainly synthesized in developing fetus and in various neoplasia, and has been associated with high microvessel density of different tumors. Materials and methods: We investigated the angiogenic activity of this protein and its possible role in the fetomaternal unit, using microvascular endothelial cells isolated from placenta and uterus in proliferation and migration assays. Pro-angiogenic activity of AFP was also studied in a spheroid in vitro angiogenic assay and in an in vivo chick chorionallantois membrane assay. Results: Physiological concentrations of AFP (10
150 ng/ml) induced proliferation of microvascular endothelial cells isolated from placenta and uterus, which could be inhibited by addition of specific antibodies against AFP or its receptor. In both proliferation as well as a spheroid in vitro angiogenic assays AFP potentiated the angiogenic activity of vascular endothelial growth factor, the key growth factor during fetal development, which could also be abolished by addition of specific antibodies against AFP or its receptor. Furthermore, AFP alone increased sprout formation in a three-dimensional microcarrier-bead in vitro angiogenic assay and induced blood vessel formation in an in vivo chick chorionallantois membrane assay. Conclusion: Our data indicate an important function of AFP as a pro-angiogenic factor during early pregnancy.

P24 Endothelium-specific Cre expression in transgenic mice directed by flk-1 gene regulatory regions Alexander Licht, Sabine Raab & Georg Breier Department of Molecular Cell Biology, Max-Planck-Institut, Bad Nauheim, Germany (E-mail: [email protected]) Background: Targeted disruption of genes is a powerful technique to study gene function in vivo. However, the analysis is often hindered by early embryonic lethality. This problem can be overcome by conditional gene ablation, leading to a cell type specific gene knock-out. The Cre-loxP system has successfully been applied to knock-out genes conditionally in mice. We recently analysed the gene regulatory regions of the mouse flk-1 gene and found that a promoter fragment in combination with a fragment of the first intron were sufficient to drive endothelium-specific expression of the LacZ reporter gene in transgenic mice. Material and methods: The flk-1 gene regulatory regions were fused to the gene encoding the Cre recombinase and transgenic mouse lines were generated by microinjection. In the resulting flk-1-Cre transgenic mice, Cre activity was determined by RT-PCR and cross-breeding with two different reporter mouse lines (Rosa26R and CAG-CAT-LacZ). In double transgenic offspring, the LacZ reporter gene is expressed only in cells in which Cre mediated recombination occurred. We examined these double transgenic mice during different

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stages of embryonic development (E9.5
E16.5), organs of adult animals and tumor neovascularisation by LacZ staining. Results: Two independent mouse lines were established which both expressed the transgene. Analysis of flk1-Cre mice crossed with reporter mice revealed a strong endothelium-specific staining of most vascular beds in embryos older than E11.5 in one or E13.5 in the second line. Also, endothelial cells in organs of adult mice were positive for LacZ. Conclusion: These results indicate that the novel transgenic mouse line flk-1-Cre can be used to delete loxPflanked target sequences efficiently in vivo. These lines will be a valuable tool for conditional gene inactivation of loxP-flanked genes in endothelial cells of the embryonic vascular system.

P25 b-Catenin is required for endothelial-mesenchymal-transdifferentiation during the formation of heart atrio-ventricular septa in the mouse Stefan Liebner, Anna Cattelino, Radiosa Gallini, Noemi Rudini, Maria Grazia Lampugnani, Giovanna Balconi, Stefano Piccolo, Rolf Kemler & Elisabetta Dejana FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy (E-mail: [email protected]) The endothelial cells (EC) b-catenin is linked to VE-cadherin at cell-to-cell adherens junctions and furthermore, it participates in wnt-signaling modulating gene transcription. Systemic deficiency of b-catenin leads to ectodermal malformation during gastrulation and early embryonic lethality. To elucidate the role of b-catenin for the vascular system, we conditionally inactivated b-catenin in ECs using the Cre/loxP system. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos, whereas later in development vascular patterning in the head, vitelline and umbilical vessels and the placenta were altered. Vessels were frequently hemorrhagic and edemas were observed in the pericardium. I addition, at 10.5 the heart showed a lack of atrio-ventricular (AV) septum formation. This process requires transdifferentiation of endocardial/endothelial cells into mesenchymal cells (EMT), which invade the acellular heart cushion. To specifically mimic this process, we utilized an established ex vivo assay, culturing AV-explants on collagen I gels. We found that in wild type (wt) explants ECs normally grew out and invaded the collagen gel, in b-catenin deficient explants (ko), ECs grew out only on the surface and failed to invade the gel. In order to elucidate if the wnt-signaling function of b-catenin is involved in the process of transdifferentiation, we investigated heart explants from animals transgenic for a wnt-signaling reporter gene based on the expression of b-galactosidase. We could demonstrate that outgrowing and some gel invading cells were positive for bgalactosidase, showing at the same time a staining for the endothelial specific marker PECAM. This strongly supports the hypothesis that endocardial b-catenin is involved in a wnt-like nuclear signaling during the process of EMT in the formation of the AV-septum. We show for the first time the role of endothelial b-catenin in vivo in a conditional knock-out approach. We could demonstrate that the absence of b-catenin in ECs leads to lack of AV-septum formation. The results obtained from ex vivo AV-explants support the idea that in the ko the process of EMT is not functional, which is at least partially due to the absence of the nuclear transcriptional function of b-catenin.

P26 PPARa activators inhibit vascular endothelial growth factor receptor-2 expression by repressing Sp1-dependent DNA binding and transactivation Markus Meissner1, Monika Stein1, Kerstin Reisinger1, Roland Kaufmann1 & Jens Gille1,2 1

Department of Dermatology, Klinikum der J.W. Goethe-Universita¨t, Frankfurt am Main, Germany; 2Department of Molecular Biology, Max-Planck-Institut fu¨r Physiologische und Klinische Forschung, Bad Nauheim, Germany (E-mail: [email protected])

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Background: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, originally implicated in the regulation of lipid and glucose homeostasis. In addition, natural and synthetic PPAR activators may control inflammatory processes by inhibition of distinct proinflammatory genes. As signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation, we explored whether known anti-inflammatory effects of PPAR ligands are mediated in part through diminished VEGFR2 expression. Material and methods: VEGFR2 gene and protein expression were analyzed in HUVECs untreated or treated with PPARa and PPARc ligands by Northern- and Western-Blots. VEGFR2 promoter
reporter gene constructs containing various deletions or mutations and gel shift assays were used to identify minimal promoter requirements and potential points of application of ligand-activated PPARs. To measure the influence of PPAR ligands on capillary-like network formation, short-time matrigel assays were established. Results: In this study, PPARa agonists are found to potently inhibit endothelial VEGFR2 expression, whereas predominant PPARc ligands remained without discernible effects. Time- and concentration-dependent inhibition is demonstrated both at the level of protein and mRNA VEGFR2 expression. In addition, VEGF-driven capillarylike network formation in Matrigel was greatly attenuated by PPARa agonists. Inhibitory effects of PPARa agonists on transcriptional activity of the VEGFR2 promoter are conveyed by an element located between bp )60 and )37 that contains two adjacent consensus Sp1 transcription factor binding sites. Constitutive Sp1/Sp3containing complex formation to this sequence is decreased by PPARa treatment. Conclusions: VEGFR2 gene expression by endothelial cells is inhibited by PPARa agonists via repressing Sp1 site dependent DNA binding and transactivation. This effect might be one of the important anti-inflammatory characteristics of PPARa ligands.

P27 Angiopoietin 2 levels in sera of children with solid malignancies Till Milde, Helena Pavlakovic & Lothar Schweigerer Department of Pediatrics I, Childrens Hospital, University of Goettingen, Germany (E-mail: [email protected]) Background: The Angiopoietins 1 and 2 (Ang1 and Ang2) mediate their effects by binding to the endothelial cellspecific receptor Tie-2. Ang1 activates Tie-2, but the exact role of Ang2 remains to be determined. Ang1 apparently contributes to the remodeling of the primary vasculature in early development by promoting circumferential growth eventually stabilizing the vasculature. In contrast, Ang2 seems to destabilize existing blood vessels rendering them more susceptible to proangiogenic signals (such as VEGF) and thereby appears to confer plasticity to the respective vessels. In the adult organism, Ang2 expression is found wherever vascular remodeling takes place, i.e. in the developing vasculature of tumors. As a secreted molecule, Ang2 should be present in serum of healthy individuals or those with malignancies. However, so far, it is unknown whether or not Ang2 is present in human serum and if so, at what concentrations. Materials and methods: Using the DuoSet ELISA Development Kit Ang2 (R& D Systems), we established an ELISA for the measurement of Angiopoietin 2 in serum. Recovery rates were in the range of 88%  11% (mean  SD). We initially determined Ang2 immunoreactivities in sera of 54 healthy children without ongoing infection or wound healing and then in 25 children with various solid malignancies including Ewing’s sarcoma, medulloblastoma, lymphoma, rhabdomyosarcoma, osteosarcoma, neuroblastoma, nephroblastoma and Wilms tumor collected at the time of diagnosis. Results: Ang2 immunoreactivities in sera of the control group ranged between 202
7685 pg/ml (mean: 590 pg/ml) as compared to Ang2 immunoreactivities between 351
9594 pg/ml (mean: 1088 pg/ml) in the patient group. The difference between controls’ and patients’ serum Ang2 immunoreactivities was statistically significant (P ¼ 0.004) as determined by Mann Whitney U Test. Conclusion: Our Ang2 ELISA is useful to further elucidate the role of Ang2 in tumor angiogenesis. The significant difference in Ang2 serum immunoreactivities in healthy children and those with solid malignancies suggest a role of Angiopoietin 2 in the progression of solid childhood malignancies.

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P28 JunB: A major player in the cellular hypoxia response? Oliver Pein1, Lars Hummerich2, Peter Lichter2, Peter Angel1 & Marina Schorpp-Kistner1 1

Deutsches Krebsforschungszentrum (DKFZ), Abteilung Signaltransduktion und Wachstumskontrolle; 2Abteilung Molekulare Genetik, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany (E-mail: [email protected])

Induction of angiogenesis and dysregulation of signal transduction pathways regulating cell proliferation and viability are hallmarks of cancer. Angiogenesis is primarily activated by VEGF, whose expression is induced by low oxygen tension (hypoxia) via the transcription factor HIF. Recently, the AP-1 member c-Jun was suggested to cooperate with HIF. Yet, gain- or loss-of-function approaches in mice did not provide supporting evidence for this assumption. By contrast, we observed several vascular and angiogenic defects in JunB-deficient embryos suggesting a critical role for JunB in the cellular response to hypoxic stress. This is supported by our preliminary data showing hypoxia-dependent transcriptional upregulation of JunB, which, in turn, participates in transcriptional regulation of c-jun, VEGF and most likely other target genes upon hypoxia. The mechanism of junB induction is presently unknown as no HIF-binding sites could be found in the junB promoter. Analysing the upstream signalling pathways mediating the hypoxia response on junB we could not collect evidence for a previously suggested involvement of ERK and JNK pathways, at least in fibroblasts. To define novel JunB target genes, which are critically involved in the hypoxic response and angiogenesis, we have used a novel and unique developmental and placental-specific cDNA clone collection from the NIA to generate DNA chips, which were subsequently used for large-scale comparative gene profiling to identify novel hypoxia-regulated genes in endothelial cells isolated from wild type and junB-/- embryos. About 560 hypoxiaregulated genes could be identified, which are regulated in a JunB-dependent or independent manner. Currently we are in the process of confirming hypoxia-dependent regulation of this subset of genes, which include e.g. transcription factors, growth factors, cell surface proteins, as well as a number of yet unknown genes, by northern blot analysis and quantitative RT-PCR in various cell types. A progress report will be given.

P29 EphB4 signaling is capable of mediating ephrinB2 induced inhibition of cell migration A. Sturz, B. Bader, K.-H. Thierauch & J. Glienke Experimental Oncology, Schering AG Berlin, Germany (E-mail: [email protected]) Background: Angiogenesis plays a major role in various pathologies. EphrinB2
EphB4 signaling has been shown to be of considerable importance for angiogenesis. It is questioned here, whether the EphB4 signal is sufficient for inhibition of migration and if the bidirectional signal into the ephrinB2 and the EphB4 exposing cell is dependent on kinase activity of EphB4 or not. Materials and methods: MVEC were prepared from human foreskin, CHO cells were stably transfected with EphB4 expressing plasmids. Cell migration experiments were performed in transwells (Costar) and Kinase activity was determined in an ELISA using MVEC or using a fusion protein containing the EphB4 kinase domain measuring 33P incorporation. Results: EphB4 phosphorylation is observed after stimulation of MVEC with ephrinB2-Fc, which can be inhibited by a preincubation of ephrinB2-Fc with EphB4-Fc. EphrinB2 alone had no effect on migration of MVEC but inhibited VEGF stimulated migration. Again the effect was blocked by preincubation with EphB4-Fc. As it is unclear which of the EphB receptors on endothelial cells is inhibitory for ephrin B2 activity, CHO-EphB4 cells were constructed and migration was stimulated by full serum. Addition of ephrinB2 lead to inhibiton of migration only in transfected cells. The inhibitory effect of ephrinB2 was blocked by the addition of small molecule EphB4 kinase inhibitors of unrelated structure and function. They did not influence migration themselves.

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Conclusion: EphrinB2 induced inhibition of cell migration is mediated directly through the activation of EphB4 and does not depend on other EphB receptors. The kinase activity ofEphB4 is pivotal for this effect.

P30 AFP-receptor: Its expression, genomic sequence and signaling pathway C. Szardening-Kirchner, O. Liang, A. Hoffmann, K.T. Preissner, U. Lang & M. Zygmunt Department of Obstetrics and Gynecology, Justus Liebig University, Klinikstrasse 32, D-35385 Giessen, Germany (E-mail: [email protected]) Angiogenesis and vascular remodeling are crucial processes in the normal development of the placenta and early events of embryo implantation as well as in tumor invasion and metastasis. AFP, a serum alpha-globulin, mainly synthesized by the fetal liver and the yolk sac, is the major carrier of polyunsaturated fatty acids during embryo-fetal development. Further studies describe AFP as associated with the regulation of growth, differentiation and regeneration. Fetal cells have the property to bind and internalize AFP. Our studies are focussed on the role of AFP in the regulation of placental vasculo- and angiogenesis and on the AFP binding sites and/or receptor (AFP-R). We investigated AFP-R expression in placenta during gestation immunhistochemically, its genomic sequence by retroviral transfection and, not yet determined, the signaling pathway with Western Blot and Flow Cytometry (FACS)-analysis. The results obtained showed (a) the expression of the AFP-receptor in the blood vessel walls from placentae of the second and third, but not of the first trimester of pregnancy, (b) a direction to a sequence on chromosome 17q22
q24, in which the cluster of placental lactogenes is located. The differential up-regulation of the AFP-receptor in endothelial cells from placenta of the second and third trimester of gestation suggests that it may play an important role in angiogenesis.

P31 Mononuclear cells in experimental autoimmune encephalomyelitis H. Wolburg1, K. Wolburg-Buchholz1,2 & B. Engelhardt2 1

Institute for Pathology, University of Tu¨bingen, Liebermeisterstrasse 8, D. 72076 Tu¨bingen, Germany; 2MaxPlanck Institute for Vascular Biology, von Esmarch Str. 56, D-48149 Mu¨nster, Germany (E-mail: hartwig. [email protected]) Background: During inflammatory diseases of the central nervous system (CNS) such as multiple sclerosis (MS) or its prototype animal model experimental autoimmune encephalomyelitis (EAE) circulating immunocompetent cells readily get access to the CNS. The way by which this is done, is a matter of debate. Most investigators believe that the inflammatory cells migrate between the endothelial cells by dissolving the tight junctions. Material and methods: Active EAE (aEAE) was induced in female SJL/N mice at the age of 10 weeks by immunization with syngeneic spinal cord homogenate. CNS tissue was fixed for electron microscopy and inflamed postcapillary venules were observed. Alternatively, tissue was prepared for immunocytochemistry using antibodies against tight junction molecules such as claudin-3 and -5, occludin and ZO-1. Results: The recruitment of immunocompetent cells across the BBB is not yet completely understood. Recruitment of leukocytes across an endothelial barrier is a complex multi-step process mediated by different adhesion molecules. The process of transendothelial migration is the least characterized. During EAE, mononuclear cells seem to take a transcellular route sparing the tight junctions. Hereby, the endothelial cells form separate spaces at the luminal surface which are re-destined as abluminal membranes by membrane remodeling, known as emperipolesis and including the remodeling of the extracelular matrix. This mode of transendothelial transmigration was not observed in the CNS yet. At the same time, modulation of tight junctions

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at the molecular, but not at the structural level, parallels transendothelial migration of mononuclear cells during CNS inflammation. Claudin-3 is selectively lost from the tight junctions of inflamed endothelium. Conclusions: The transendothelial transmigration of mononuclear cells apart from the paracellular way controled by the tight junctions is a novel finding and a challenge for further molecular analysis. The complex alteration of endothelial shaping during transmigration may be a response to altered leukocyte-endothelial as well as endothelial-pericytic interactions. The involvement of tight junction modulation in the transcellular migration is not understood and requires further elucidation of endothelial signaling processes.

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The Endothelium in Atherosclerosis Saturday, July 19, 2003, 10.50
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P32 Hydrogen peroxide induces E-selectin in human endothelial cells independent of nuclear factor-jB activation Wolfgang Erl1,2, Go¨ran K. Hansson2 & Guro Valen3 1

Institut fu¨r Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany; 2Center for Molecular Medicine, Stockholm, Sweden; 3Crafoord Laboratory of Experimental/Thoracic Surgery, Karolinska Hospital, Stockholm, Sweden (E-mail: [email protected])

Background: Reactive oxygen species may be inflammatory signal mediators. We investigated if H2O2 induces expression of adhesion molecules and adhesion of monocytic cells in human umbilical vein endothelial cells (HUVEC). Material and methods: Detection of mRNA was by semi-quantitative RT-PCR; measurement of adhesion molecule expression was by flow cytometry (FACS), activation of transcription factors AP-1 or NF-jB by electrophoretic mobility shift assay (EMSA). We used PD098059 (PD) and SB203580 (SB), specific inhibitors of the MAP kinase pathway, or MG132, an inhibitor of NF-jB. Adhesion assays were performed with 30 min coincubation of HUVEC and monocytic U937 cells. Results: After stimulation with 200 lM H2O2, mRNA for ICAM-1 and E-selectin was increased, with a maximum at 6 h. FACS analysis demonstrated increased E-selectin and ICAM-1 protein, but no VCAM-1. H2O2 enhanced E-selectin-dependent adhesion of U937 cells to HUVEC, indicating upregulation of functional adhesion molecules. PD and SB reduced H2O2-induced E-selectin and ICAM-1 protein expression, and adhesion of U937 cells to HUVEC. H2O2 activated AP-1, but not NF-jB, while TNF-a (10 U/ml) induced both, and increased the expression of all three adhesion molecules tested. Conclusion: H2O2 induces MAP kinase-dependent AP-1 activation, which leads to expression of E-selectin, and increased adhesion of monocytes to endothelium. This mechanism may play a role in monocyte recruitment in conditions such as inflammation, re-perfusion, and atherosclerosis.

P33 Nucleo-cytoplasmic shuttling of LIMK-2 in endothelial cells Pankaj Goyal1, Dharmendra Pandey2 & Wolfgang Siess1 1

Institut fu¨r Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig Maximilians Universita¨t, Mu¨nchen, Germany (E-mail: [email protected])

Background: Various stimuli like LPA, thrombin induce endothelial cell shape change via Rho/Rho-kinase mediated reorganization of the actin cytoskeleton. LIM-kinases regulate actin cytoskeletal reorganization through phosphorylation of cofilin at Ser-3. The LIMK family kinases, composed of LIMK-1 and LIMK-2, possess characteristic structural features, consisting of two LIM domains and a PDZ domain in the N- terminal half, and a kinase domain in the C-terminal half. We are investigating the role of LIMK-2 for cofilin phosphorylation and actin dynamics in endothelial cells. Methods: Endothelial cell cDNA was synthesized and analyzed by RT-PCR. Transfection of endothelial cells with various GFP constructs of LIMK-2 and YFP-b-actin plasmid was done by electroporation. Fluorescence live imaging, fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photo-bleaching (FRAP) analysis were done by confocal microscopy.

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Results: In live imaging of sub-confluent endothelial cells using YFP-b-actin, we found that thrombin stimulated the contraction of actin stress fibers, cell rounding, and membrane ruffling. By RT-PCR we found that LIMK-2 is present in endothelial cells. The GFP tagged C-terminal half of LIMK-2 (314
638), containing the kinase domain in endothelial cells was localized mainly in the nucleus, and also in the cytoplasm with very little on stress fibres. It has one nuclear localization signal (NLS 479
503) and one nuclear export signal (NES 604
615). By FLIP and FRAP analysis we observed a rapid shuttling between the nucleus and the cytoplasm. The DLIM LIMK-2 (142
638) construct containing the PDZ and the kinase domain was enriched in the nucleus and associated with the stress fibres. It has a second NES (240
249) between the PDZ and the kinase domain. Full length LIMK-2 was only in the cytoplasm. However, it was exclusively present in the nucleus, when the cells were treated with Leptomycin B (LMB), a specific inhibitor of CRM-1 dependent nuclear export. Conclusions: In sub-confluent endothelial cells, LIMK-2 is rapidly shuttling between the nucleus and the cytoplasm as unravelled by inhibition of the nuclear exporter CRM1. LIM domains of LIMK-2 drive the CRM-1 dependent nuclear export of LIMK-2 probably by masking the NLS. The PDZ domain might be required for the association of LIMK-2 with the stress fibres, whereas LIM domains seem to inhibit this association.

P34 Rhoa and Rac1 mediate uPA-induced migration of VSMC I. Kiian, N. Tkachuk, H. Haller & I. Dumler Department of Nephrology, Hannover Medical School, Hannover, Germany (E-mail: kiian.ioulia@ mh-hannover.de) Background: Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) mediate migration of VSMC and can contribute to atherosclerotic process. uPA/uPAR signaling required for VSMC migration is mediated by Tyk2/PI3-K pathway. We investigated downstream components of uPAR induced pathway in VSMC. Material and methods: Association of proteins was assessed with affinity precipitation and immunoprecipitation. Activation of Rho proteins was estimated with pull-down assay. Cell migration was measured in Boyden chamber and by microscopy after microinjection with inhibitory antibody. Redistribution of GTP-RhoA and GTPRac1 was observed after immunocytochemical staining of VSMC using GST fusion proteins with Rho-binding domains of RhoA and Rac1 effector proteins. Results: We report that active forms of small GTPases RhoA and Rac1 were associated with uPAR/Tyk2/PI3K signaling complex. RhoA was transiently and significantly activated in response to uPA in a PI3-K-dependent manner. Downstream RhoA, Rho kinase upregulation led to increased phosphorylation of myosin light chain (MLC). Rho kinase inhibition decreased both, uPA-stimulated MLC phosphorylation and VSMC migration as was observed in Boyden chamber. Neither Rac1 nor Cdc42 was activated in response to uPA. Nevertheless, Rac1 turned out to be also required for VSMC migration towards uPA since its inhibition led to decreased cell migration. VSMC immunocytochemical staining demonstrated localization of activated RhoA and Rac1 to the newly formed leading edge of VSMC migrating towards uPA. Conclusion: Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary mediators the uPA/uPAR-directed migration via the Tyk2/PI3-K signaling complex in human VSMC.

P35 Niacin stimulates the transcription of reverse cholesterol transporter ABCA1 in endothelial and monocytoid cells Tina Rubic, Matthias Trottmann & Reinhard Lorenz Institut fu¨r Prophylaxe der Kreislaufkrankheiten, LMU Muenchen, Germany (E-mail: [email protected]) Background: Niacin, the first lipid lowering drug shown to improve survival after myocardial infarction, decreases LDL and increases HDL cholesterol levels. These effects cannot fully be explained by its suspected mechanism

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of action, inhibition of lipolysis and hepatic VLDL synthesis. Niacin has also been shown to massively stimulate prostaglandin D2 (PGD2) formation. 15d-PGJ2, the major PGD2 metabolite, activates PPARc and can enhance ABCA1 and CD36 expression. We therefore studied the effects of niacin on these key receptors of reverse cholesterol transport. Material and methods: The human endothelial cell line EA.hy 926 and the monocytoid cell line Mono Mac 6 (MM6) were incubated with 1000 lM niacin or carrier control for 3
72 h. The transcription of ABCA1, CD36 and LDL-R was quantified with real-time RT-PCR. The PPARc protein expression was measured by flow cytometry and PPARc translocation by western blot of cytosolic and nuclear extracts. To characterise the effects on cholesterol handling an oxidised LDL uptake/HDL driven efflux assay was used. Results: Niacin stimulated the ABCA1 transcription in endothelial and monocytoid cells. The transcription and protein expression of PPARc and CD36 in MM6 cells was increased and PPARc nuclear translocation was enhanced. The LDL-receptor (LDL-R) remained unchanged. Thereby niacin enhanced HDL mediated cholesterol efflux from the cells resulting in a reduced cellular cholesterol content. The niacin effect on CD36 was prevented by cyclooxygenase inhibition, whereas the niacin effect on ABCA1 was only prevented by protein kinase A (PKA) inhibition and mimicked by a cyclic AMP (cAMP) analogue, suggesting mediation by the 15dPGJ2/PPARc and the cAMP/PKA pathways, respectively. Conclusion: These new actions of niacin on several key effectors of reverse cholesterol transport out of the vessel wall provide a rational to test niacin for regression of atherosclerosis and the combination of niacin with statins for an overadditive clinical benefit independent of its action on LDL-levels.

P36 Stabilization of atherosclerotic plaque by blockade of macrophage migration inhibitory factor after arterial injury in apolipoprotein E-deficient mice Andreas Schober, Ju¨rgen Bernhagen, Sandra Knarren, Richard Bucala & Christian Weber Molecular Cardiovascular Research, University Hospital, Aachen, Germany (E-mail: [email protected]) Macrophage migration inhibitory factor (MIF), a cytokine that controls cell-mediated inflammatory responses, is upregulated in early atherogenesis and during the progression of atherosclerosis; however its functional contribution to lesion development has not been evaluated. We studied the effect of a blocking anti-MIF monoclonal antibody (MIF mAb) on neointima lesion formation following wire-induced carotid injury in apolipoprotein E-deficient (ApoE-/-) mice. Either MIF mAb or a control mAb was injected twice weekly (100 lg per injection) up to 3 weeks after carotid injury. Neointimal plaque volume was determined in Movats pentachrome stained serial sections of the common carotid artery. Neointimal macrophage and smooth muscle cell (SMC) content was evaluated by quantitative immunohistochemistry using Mac-2- and SMC-specific a-actin Ab. Cellular MIF expression was studied by immunostaining. Serum samples were analyzed for lipids. Flow chamber assays were performed using monocytic cell line perfused on human aortic endothelial cells with or without MIF pretreatment. Treatment with MIF mAb did not affect lipid serum levels. Immunohistochemistry revealed endothelial MIF staining in ApoE-/- mice on a high cholesterol diet. As early as 24 h after endothelial denudation MIF was detectable in SMC. Double immunofluorescence staining confirmed MIF expression in neointimal SMC and macrophages 2 weeks after injury. Reendothelialized plaque areas showed strong endothelial MIF expression. Notably, the blockade of MIF resulted in a 50% reduction of macrophage content in neointimal lesions (26%  6.3% vs. 52%  6.4%, n ¼ 5, P < 0.02). Concomitantly, the neointimal content of SMC was increased in ApoE-/- mice treated with MIF mAb (44%  4% vs. 24%  1.5%, n ¼ 5, P < 0.001). Differences in total plaque volume between MIF and control mAb-treated ApoE-/- mice were not significant. In vitro flow assays revealed that MIF immobilized on unstimulated human aortic endothelial cells effectively triggered adhesion of monocytic cells. Inhibition of MIF resulted in a shift of the cellular composition of atherosclerotic plaques towards a more stabilized phenotype with reduced macrophage/foam cell content, likely due to reduced MIF dependent monocyte recruitment on endothelium.

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P37 Enhanced platelet adhesion and platelet secretion in vasodilator-stimulated phosphoprotein deficient mice Christian Schulz1, Steffen Massberg1, Sabine Gru¨ner1, Ildiko Konrad1, Martin Eigenthaler2, Ulrich Walter2 & Meinrad Gawaz1 1

Deutsches Herzzentrum und 1. Medizinische Klinik, Technische Universita¨t Mu¨nchen, Germany; 2Institut fu¨r Klinische Biochemie und Pathobiochemie, Julius-Maximilians-Universita¨t Wu¨rzburg, Germany (E-mail: [email protected])

Background: Platelet adhesion and activation at the vascular wall are the initial steps leading to arterial thrombosis and vascular occlusion. Prostacyclin and nitric oxide inhibit platelet adhesion, acting via cAMP- and cGMP-dependent protein kinases. A major downstream target for both cAMP- and cGMP-dependent protein kinases is the vasodilator-stimulated phosphoprotein (VASP), a cytoskeleton- and integrin-associated platelet protein. Methods and results: To test the significance of VASP for the regulation of platelet adhesion in vivo, we studied platelet-vessel wall interactions using VASP-deficient (VASP-/-) mice. Under physiological conditions, platelet adhesion to endothelial cells was significantly enhanced in VASP null mutants, when compared to wild type mice (P < 0.05). The loss of VASP also increased platelet adhesion to the postischemic intestinal microvasculature, to the atherosklerotic endothelium of ApoE-deficient mice, and to the subendothelial matrix following endothelial denudation (P < 0.05 vs. wild type). Further, increased platelet adhesion in VASP null mutants was completely unresponsive to preincubation with the nitric oxide donor spermine-NO. Finally, measurement of static adhesion to fibrinogen and collagen in vitro showed an enhanced a-granule secretion of platelets taken from VASP-/- mice (P < 0.05 vs. wild type platelets). Conclusion: These data show for the first time in vivo that VASP acts as a negative regulator of platelet adhesion to the vascular wall under both physiological and pathophysiological conditions and point to the importance of VASP as a target for novel anti-platelet therapies.

P38 Effects of isoprostane 8 on human endothelial cells in vitro A. Tacheny, M. van Steenbrugge, D. Colette, C. Gustin & M. Raes URBC, FUNDP, University Namur, Namur, Belgium (E-mail: [email protected]) Background: Increased isoprostane levels were found in patients with atherosclerotic lesions. In this study, we investigated the effects of isoprostane 8 (8-iso-PGF2a) on HUV-EC-C cells cultured in vitro, with a particular focus on the signal transduction pathways involved. Material and methods: The effects of 8-iso-PGF2a were analysed on transcription factor activation (NFjB and AP-1) using a colorimetric assay (Renard et al., Nucleic Acids Res. 2001: 29: 1
5), on MAPkinase phosphorylation (using immunocytochemistry coupled to confocal microscopy and Western Blot), on MCP-1 protein expression through an ELISA kit and on the adherence of THP-1 monocytic cells on EC (by an adherence microplate assay). This study was also completed with a proteomic approach on 2D gels to define the set of particular spots differentially expressed after 8-iso-PGF2a treatment and with a transcriptomic approach by hybridisation on a ‘medium density’ DNA microarray (about 200 genes involved in cell metabolism, cell proliferation, adherence, mobility, defense,. . .). Results: At the concentration used (10 lM), 8-iso-PGF2a activates the transcription factor AP-1 (300% of the negative control). Nevertheless, 8-iso-PGF2a seemed unable to activate NFjB. Phosphorylation of both ERK 1/2 and p38 was increased by 8-iso-PGF2a, which is in agreement with the observed activation of AP-1. Finally, 8iso-PGF2a induced a weak but reproducible increase of MCP-1 expression and adherence of THP-1 cells on EC,

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compared to TNFa. We are presently comparing gene expression at the mRNA level in unstimulated cells and in cells stimulated with 8-iso-PGF2a, TNFa (positive control) or U46619 (a TXA2 receptor agonist). Regarding the proteomic approach, preliminary electrophoretic separations on 2D gels have been run, but the comparative analysis is still underway. Conclusion: Our results (MAPK phosphorylation and AP-1 activation, MCP-1 over-expression and increased adherence of monocytic cells), favour the implication of 8-iso-PGF2a in the early steps of atherosclerosis as a factor able to activate EC. Further investigations are required.

Acknowledgements This poster presents research results of the Belgian Programme on IAP initiated by the Belgian State, Primer Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by its authors. Aure´lie Tacheny is recipient of a FRIA fellowship.

P39 4-Hydroxynonenal induces oxidative stress and apoptosis in human endothelial cells Claudia A. Vosseler, Peter C. Weber & Wolfgang Erl Institut fuer Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-University, Munich, Germany (E-mail: [email protected]) Background: Injury of the vascular endothelium is a critical step in the development of atherosclerosis and is at least in part mediated by inflammatory and oxidative mechanisms. Lipid peroxidation products such as oxLDL have been detected in atherosclerotic lesions. OxLDL is a complex mixture of various components including 4hydroxynonenal (4-HNE). 4-HNE is a reactive aldehyde which has been shown to induce oxidative stress in various cell types. We therefore investigated its effects on human endothelial cells with emphasis on a possible role in the induction of apoptosis. Material and methods: HUVECs were isolated from human umbilical cord veins and cultured in endothelial cell growth medium. 4-HNE was dissolved in ethanol (max. conc. 0.1%). Confluent HUVECs were treated with 0
50 lM 4-HNE. Cell viability was determined by MTT assay or trypan blue exclusion. Apoptosis detection was performed by doublestaining of cells with Annexin V-FITC and PI. Hydroethidine staining was used to detect reactive oxygen species. Both analyses were done on a FACScan using Cell Quest software. To determine oxidative stress, GSH content was measured photometrically at 405 nm. Results: Incubation of HUVECs with 4-HNE induced a time- and dose-dependent decrease in cell viability and cell number as shown by MTT assay and trypan blue exclusion. Whereas higher doses of 4-HNE (30
50 lM) seemed to be cytotoxic, a reduction in cell viability and number by 10 and 20 lM 4-HNE was due to induction of apoptosis as shown by Annexin V-staining. Hydroethidine staining demonstrated that 4-HNE treatment of HUVECs induced the formation of reactive oxygen species. This was accompanied by a reduction of cellular GSH content, reaching a minimum after approximately 2 h. In surviving cells a subsequent up-regulation of GSH was observed. Conclusion: Our results show, that 4-HNE markedly reduces HUVEC viability and cell number. Low concentrations induce apoptosis whereas higher concentrations (>30 lM) possibly induce necrotic cell death. The predominant underlying mechanism seems to be a reduction in GSH content and formation of reactive oxygen species.

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P40 Stereospecific and redox-sensitive increase in monocyte adhesion to endothelial cells by homocysteine O. Postea1, F. Kroetz2, S. Hillebrand1, B. Banas1, S. Zahler2, C. Keller1 & N. Weiss1 1

Medizinische Poliklinik
Innenstadt Mu¨nchen, Germany; Germany (E-mail: [email protected])

2

Institut fu¨r Physiologie, Universita¨t Mu¨nchen,

Background and aims: Hyperhomocysteinemia induces endothelial dysfunction and promotes atherosclerotic vascular disease. As the adhesion of mononuclear cells (MO) to activated/dysfunctional endothelial cells (EC) is crucial for the development of atherosclerosis, we investigated the effect of homocysteine (Hcy) on the interaction between cultured EC (EA.hy 926 cells) and MO (THP-1 cells). Methods: Adhesion of fluorescence-labelled MO to EC, incubated with different Hcy concentrations in the pathophysiological relevant range (10
200 lM) and with different Hcy species (D,L-Hcy, L-Hcy, D-Hcy), and with antioxidants (superoxide dismutase mimetic complex, MnTBAP) and blocking antibodies, was studied under static coculture conditions. Expression of adhesion molecules on EC was studied by FACS-analysis. Intracellular reactive oxygen species (ROS) were measured using the redox-sensitive dye, DCF-DA, and quantified by image analysis during confocal microscopy. Results: Incubation of EC with D,L-Hcy and L-Hcy dose-dependently and significantly increases MO-adhesion to EC up to fivefold compared to control. In contrast, D-Hcy and L-cysteine has no significant effect. The increase in MO adhesion to Hcy-incubated EC is paralleled by an increase in DCF-fluorescence in D,L- and L-Hcy-treated EC, indicating increased formation of ROS in Hcy-incubated EC. In accordance, the increase in MO-adhesion is completely abolished by coincubation of EC with D,L-Hcy and the superoxide dismutase mimetic complex, MnTBAP. FACS-analysis shows a significant increase in ICAM-1 expression on Hcy-treated EC. In accordance, pretreatment of EC with an anti-ICAM monoclonal antibody reduces MO-adhesion to Hcy-incubated EC, suggesting that Hcy may influence monocyte-EC interaction through the modulation of ICAM-1 expression. Conclusions: Vascular oxidant stress in hyperhomocysteinemia might promote the development of atherosclerotic lesions by inducing inflammatory changes in EC that lead to adhesion of circulating leukocytes. Antioxidants might have a role in preventing Hcy-induced vascular disease.

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The Endothelium in Inflammation Saturday, July 19, 2003, 14.00
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P41 PPAR-gamma-independent induction of endothelial cell apoptosis by cyclopentenone prostaglandins Wolfgang Erl, Alma Zernecke, Christian Weber & Peter C. Weber Institut fu¨r Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany (E-mail: [email protected]) Background: Cyclopentenone prostaglandins (CP-PGs), such as prostaglandin A1 (PGA1) or 15-deoxy-D12,14prostaglandin J2 (PGJ2), have been proposed to exert anti-inflammatory effects in macrophages and smooth muscle cells, which may be mediated by activation of the peroxisome-proliferator activated receptor-c (PPAR-c). In endothelial cells, PGJ2 has been shown to inhibit angiogenesis. PGJ2 induces apoptosis in different cell types. Although CP-PGs have been studied in endothelial cells, their effects on endothelial cell apoptosis are not clear. Material and methods: We used cultures of human umbilical vein endothelial cells (HUVEC). Cell viability was determined by trypan blue assay. Apoptosis was characterized by Annexin V binding and a sub-G1 peak in the cell cycle, measured with a FACScan. The expression of apoptosis proteins was detected by intracellular staining and analyzed by flow cytometry. Results: Incubation with PGA1 or PGJ2 for 24 h reduced HUVEC number and viability. PGA1 or PGJ2 induced phosphatidylserin externalization, and activated caspase-3, resulting in an increased percentage of cells with a DNA content <2N. Furthermore, CP-PG treatment reduced the expression of the anti-apoptotic proteins bcl-2 and iap-1. Pre-incubation of HUVEC with the pan-caspase inhibitor z-VAD-fmk prevented apoptosis and CP-PGinduced PPAR-c-degradation. The use of PPAR-c-deficient embryonic stem cells, differentiated into endotheliallike cells, enabled us to demonstrate that the induction of apoptosis in endothelial cells by CP-PGs is independent of PPAR-c activation. Conclusion: The findings show that CP-PGs induce apoptosis in HUVEC independently of PPAR-c. At sites of inflammation, the synthesis of CP-PGs, such as PGJ2, is increased. This could be a possible mechanisms for endothelial cell damage and the initiation of atherosclerotic lesions.

P42 Morphology of the endothelial glycocalyx in the coronary vasculature after the infusion of clinically used colloids and after short ischemia M. Lo¨tsch1, M. Rehm2, S. Zahler1, U. Welsch3, P. Conzen2, M. Jacob2 & B.F. Becker1 1

Department of Physiology; 2Clinic of Anaesthesiology; 2Department of Anatomy; Ludwig-Maximilians-University of Munich, Munich, Germany (E-mail: [email protected]) Background: Based on a meta-analysis of haemodilution studies and supplementary experimental data, Pries et al. concluded that the dilution of blood with artificial media in form of infusion solutions can induce a dissolution of the absorbed layer of plasma proteins from the endothelial glycocalyx, thereby modifying the morphology and function of the glycocalyx [1]. Also 30
90 min periods of ischemia resulted in damage to the endothelial glycocalyx [2]. Using an isolated heart preparation and electron microscopy, we evaluated the changes in the morphology of the endothelial glycocalyx after the infusion of commonly used colloids and a short period of ischemia.

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Materials and methods: Guinea-pig hearts were isolated and perfused with a modified Krebs
Henseleit buffer in Langendorff-mode. In order to administer infusion solutions, a small catheter, connected with an infusion pump, was inserted into the aortic feed line. After an equilibration phase of 15 min, solutions of either 5% human albumin or 6% hydroxyethylstarch [molecular weight 200 kD/grade of substitution 0.5] were infused continuously for 20 min at a rate constantly adjusted to 1/3 of the actual coronary flow. The same protocols were performed with the inclusion of 15 min of global ischemia between equilibration and infusion of colloid solutions. After the infusion protocol, hearts were perfusion-fixed and electron microscopy was performed in modification of a method described by Vogel et al. [3], using lanthanum nitrate for staining the endothelial glycocalyx. Results: Electron microscopy revealed an endothelial glycocalyx with a thickness of about 200
300 nm in buffer-perfused hearts. Exactly the same thickness of glycocalyx was apparent in hearts after the infusion of human albumin or hydroxyethylstarch solutions. Also 15 min of ischemia resulted in no significant damage to the glycocalyx, irrespective of whether colloids were present during perfusion or not. Conclusion: Clinically used colloid solutions seem to have no impact on the morphology of the endothelial glycocalyx in the intact coronary system, neither alone nor in conjunction with a short period of ischemia. References [1] Pries AR, Secomb TW, Gaehtgens P. Blood flow resistance during hemodilution: Effect on plasma composition. Cardiovasc Res 1998; 37: 225
35. [2] Ward BJ, Donnelli JL. Hypoxia-induced disruption of the cardiac endothelial glycocalyx: Implications for capillary permeability. Cardiovasc Res 1993; 27: 384
9. [3] Vogel J, Speradino M, Pries AR, Linderkamp O, Gaehtgens P, Kuschinsky W. Influence of the endothelial glycocalyx on cerebral blood flow in mice. J Cereb Blood Flow Metab 2000; 20: 1571
8.

P43 A novel cRel/ISRE promoter module triggers GBP-1 expression in endothelial cells in response to inflammatory cytokines Elisabeth Naschberger, Thomas Werner, Ana B. Vicente, Eric Guenzi, Clara Lubeseder-Martellato, Kristin To¨polt, Peter J. Nelson & Michael Stu¨rzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, 85764 Neuherberg, Germany (E-mail: [email protected]) Background: The guanylate binding protein (GBP)-1 characterizes endothelial cells activated by inflammatory cytokines (IC) such as IFN-c, IL-1b and TNF-a and mediates the anti-proliferative effect of IC in these cells. Induction of GBP-1 expression by IFN-c is mediated via an IFN-a-stimulated response element (ISRE) and an IFN-c activation site (GAS). We investigated which promoter elements may trigger GBP-1 expression in HUVEC in response to IL-1b and TNF-a. Material and methods: IC-induced GBP-1 promoter activity was analyzed by transfection experiments in HUVEC and detection of the firefly luciferase reporter gene activity. Potential transcription factor binding sites were predicted by computational analysis (MatInspector and Genomatix Suite, Genomatix). The binding site analysis was verified by transfection experiments with mutated promoter constructs and by electrophoretic mobility shift assay (EMSA). Results: IL-1b and TNF-a in combination with IFN-c increased GBP-1 expression in HUVEC synergistically, suggesting the involvement of a second element different from ISRE/GAS. We detected a cRel element cooperating with ISRE in the regulation of IC-induced GBP-1 expression in a module like manner. Induction of GBP-1 expression by IL-1b and TNF-a required both elements, whereas ISRE was sufficient for a transcriptional response to IFN-c. Concordantly, ISRE was activated by all three IC via IRF-1 binding. The cRel site was selectively activated by IL-1b and TNF-a via specific NF-jB heterodimers (p65/p50). Detection of the cRel/ISRE module in different inflammation-associated genes supported its function as an IC-response module. Conclusion: IC-induced GBP-1 expression is mediated via a novel cRel/ISRE promoter module that regulates the synergistic activity of IL-1b and TNF-a in combination with IFN-c in primary endothelial cells. The cRel/ISRE promoter module described here may define a first member of a family of IC-response modules that may regulate gene expression in inflammatory diseases.

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P44 Treatment of human endothelial cells with the inflammation-inducing agent cobalt chloride leads to cell cycle inhibition that is not mediated by guanylate binding protein-1 (GBP-1) K. Peters1, A. Scholz1, C. Lubeseder-Martellato2, M. Stu¨rzl2, R.E. Unger1 & C.J. Kirkpatrick1 1

Institute of Pathology, Langenbeckstr. 1, 55101 Mainz, Germany; 2Institute of Molecular Virology, Department of Virus-induced Vasculopathy, Ingolsta¨dter Landstr. 1, 85764 Neuherberg, Germany (E-mail: peters@ pathologie.klinik.uni-mainz.de) The implantation of cobalt-chromium alloys as bone substitutes can lead to high concentrations of divalent cobalt ions in the peri-implant tissues. Whether the presence of such corrosion products leads to complications in implant integration and long-term stability is unknown. The exposure of endothelial cells to divalent cobalt ions leads to an inflammatory response. This is characterized by the release of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1). This cytokine release is mediated by the activation of transcription factor NF-jB. The stimulation of human endothelial cells with pro-inflammatory cytokines (TNFa, IFNc) induces the expression of the guanylate-binding protein-1 (GBP-1). During cytokine induced inflammation an inhibition of endothelial cell proliferation occurs. This inhibition of proliferative activity is shown to be mediated by GBP-1. This study examines the effects that divalent ions have on endothelial cell proliferation. Here, we could show that cobalt ion concentrations of 0.7 mM, a concentration lower than detected in peri-implant tissues, lead to a strong reduction of cell number and Ki67-expression (antigen indicating cell cycle affiliation) and an arrest in cell cycle. Thus, the cobalt exposure induces effects similar to those observed by pro-inflammatory cytokines (proinflammatory activation and inhibition of proliferative activity). Therefore we tested the expression of GBP-1 in cobalt-treated human endothelial cells. Here, we could show that the expression of GBP-1 is not enhanced compared to the untreated controls. Therefore, cobalt chloride-treatment of human endothelial cells leads to the pro-inflammatory activation and an arrest in cell cycle progression without the involvement of GBP-1. If an inhibition of endothelial cell proliferation induced by cobalt ions also occurs in the peri-implant tissues, then an impairment of wound healing (involving blood vessel formation etc.) and reduced implant stability could be a possible consequence. This work is supported by the Deutsche Forschungsgemeinschaft.

P45 Human neutrophils promote angiogenesis: A putative role for endothelial interleukin 8 Ruth Schru¨fer1, Nicola Lutze2, Ju¨rgen Schymeinsky1 & Barbara Walzog1 1

Department of Physiology, Ludwig-Maximilians-Universita¨t Mu¨nchen, Mu¨nchen; Berlin, Germany (E-mail: [email protected])

2

Freie Universita¨t Berlin,

Background: Human polymorphonuclear neutrophils (PMN) play an important role in host defence and inflammation. In the present study, we investigated the question whether PMN may also contribute to the induction of inflammation-mediated angiogenesis. Methods: Angiogenesis was measured in a three-dimensional fibrin gel using microbeads coated with human umbilical vein endothelial cells (HUVEC). Angiogenesis was induced by addition of the supernatant obtained from isolated human PMN which were stimulated by 100 nM N-formyl-Met-Leu-Phe (fMLP). Expression of interleukin 8 (IL-8) und vascular endothelial growth factor (VEGF) was analysed by semi-quantitative RT-PCR at the mRNA level and by ELISA technique at the protein level.

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Results: We found that fMLP-activated human PMN released a pro-angiogenic entity within 1 h after stimulation which induced sprouting of capillary-like structures at day 3 after the onset of the angiogenesis assay. The effect was comparable to the pro-angiogenic activity of 100 ng/ml of VEGF or basic fibroblast growth factor (bFGF). By means of ELISA technique, we found that activated PMN released the pro-angiogenic factors VEGF and IL-8: Within 1 h after the onset of stimulation, the supernatant of fMLP-activated PMN (5 · 106/ml) contained 80 pg/ml of IL-8 and 40 pg/ml of VEGF. Whereas the release of IL-8 was almost completely blocked by actinomycin D or cycloheximide, the inhibition of mRNA or protein synthesis had no effect on the release of VEGF. This suggested that the pro-angiogenic potential of PMN consists of at least two components: The de novo synthesis of IL-8 as well as the release of preformed VEGF. This was confirmed by RT-PCR which showed that IL-8 mRNA was up-regulated upon PMN stimulation whereas the expression of the VEGF mRNA was not affected. Furthermore, the pro-angiogenic effect of the supernatant obtained from activated PMN was almost completely inhibited in the presence of neutralising antibodies against VEGF or IL-8 demonstrating an important role for IL-8 and VEGF in our assay system. However, exogenous IL-8 alone failed to induce angiogenesis suggesting that IL-8 is required but not sufficient to induce angiogenesis. Finally, we studied the putative role of endothelial IL-8 and found a strong up-regulation of the endothelial IL-8 mRNA upon stimulation of HUVEC by the supernatant obtained from activated PMN. Conclusion: The present study suggests that activated PMN promote angiogenesis by releasing VEGF which may activate a paracrine feedback mechanism involving endothelial IL-8. Supported by DFG (WA 1048/1-1).

P46 Fractalkine mediates platelet activation and adhesion Christian Schulz1, Andreas Scha¨fer2, Moritz Stolla1, Iris Mu¨ller1, Steffen Massberg1, Johann Bauersachs2 & Meinrad Gawaz1 1 2

Deutsches Herzzentrum und 1. Medizinische Klinik, Technische Universita¨t Mu¨nchen, Germany; Medizinische Klinik der Julius-Maximilians-Universita¨t Wu¨rzburg, Germany (E-mail: [email protected])

Background: Fractalkine (CX3CL1), a recently discovered membrane-bound chemokine with a transmembrane domain, is expressed on endothelial cells following vascular injury and during atherosclerosis. Further, fractalkine has been described to mediate leukocyte adhesion to the endothelium via its specific receptor CX3CR1. Since several chemokines released by the endothelium also have pro-aggregatory properties, the aim of our present study was to assess the effects of fractalkine on platelet function. Methods and results: First we addressed surface expression of fractalkine on cultured human endothelial cells. While fractalkine expression was found to be low on resting endothelial cells, surface expression was upregulated by the inflammatory agent TNF-a, as demonstrated by flow cytometric studies and western blot technique. We than determined the role of fractalkine in the regulation of platelet secretion. Blood was taken from healthy human individuals and surface-expression of P-selectin, a marker of platelet a-granule secretion, was assessed by flow cytometry. P-selectin expression was significantly enhanced by in vitro stimulation with human fractalkine when combined with low levels of ADP as compared to ADP in the absence of fractalkine. Selective inhibition of fractalkine by an antagonising antibody or the disruption of the G-protein coupled intracellular signalling cascade of the fractalkine receptor by pertussis toxin (PTX) prevented platelet activation. Next we addressed the effect of fractalkine on platelet adhesion under shear conditions using a flow chamber model. Stimulation with fractalkine significantly enhanced platelet adhesion to collagen and fibrinogen but not to BSA. Similar to P-selectin expression, enhanced adhesion could be prevented by the antagonizing antibody or preincubation of platelets with PTX. Conclusion: Fractalkine, a recently discovered membrane-bound chemokine, is overexpressed on endothelial cells under inflammatory conditions. Further, fractalkine contributes to increased platelet activation and adhesion possibly modulating platelet-endothelial cell interaction. Hence, fractalkine might play an important role in increased thrombogenesis in vascular injury and atherosclerosis.

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P47 Structure-function analysis of the anti-proliferative activity of GBP-1 in endothelial cells Kristin To¨polt, Eric Guenzi, Clara Lubeseder-Martellato, Anita Jo¨rg, Elisabeth Naschberger, Andreas Stampfl & Michael Stu¨rzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, 85764 Neuherberg, Germany (E-mail: [email protected]) Background: Inflammatory cytokines (IC) inhibit endothelial cell (EC) proliferation whereas angiogenic growth factors (AGF) activate proliferation and migration of EC. Recently we identified the large GTPase human guanylate binding protein -1 (GBP-1) as specific mediator of the anti-proliferative effect of IC on EC. At the structural level GBP-1 exhibits a globular a,b-domain with GTPase activity and a purely a-helical domain, which carries a CaaX-isoprenylation motif. Material and methods: The effect of GBP-1 on major signal transduction pathways and apoptosis in retrovirally transduced HUVEC constitutively expressing GBP-1 was investigated by immunostaining of single cells and Western blot analyses. HUVEC expressing truncated forms of GBP-1 were analysed in cell counting experiments to determine the minimal domain of GBP-1 involved in the inhibition of EC proliferation. Tube forming capability of GBP-1 expressing HUVEC was investigated in a matrigel assay. Results: We could demonstrate that, the inhibition of EC proliferation by GBP-1 is independent of its GTPase activity or isoprenylation, but is mediated by a region of 166 amino acids within the a-helical domain. GBP-1 inhibited EC proliferation without inducing apoptosis, affecting morphogenesis or activation of the MAPK ERK1/ 2, p38, SAPK/JNK or the transcription factor NF-jB. Interestingly, GBP-1 expression increases the basal level of the free intracellular Ca2+ and decreases the Ca2+ release from intracellular stores in histamine stimulated cells. Conclusion: GBP-1-derived peptide sequences may be useful to inhibit endothelial cells proliferation during tumour angiogenesis whereas inhibition of GBP-1 expression might be of therapeutic interest in inflammatory diseases and to support collateral vessel formation in ischemia. Moreover, GBP-1 may exert its antiproliferative activity via the Ca2+ signal transduction pathway.

P48 Short term effects of menadione on the paracellular permeability of a brain endothelial cell monolayer K. Voigt1,2, J. Kraus1, P. Oschmann2, B. Engelhardt1 & M. Clauss1 1

MPI for Physiol. and Clin. Research, Bad Nauheim, Giessen, Germany; 2Department of Neurology, University Hospital Giessen, Giessen, Germany (E-mail: [email protected])

Background: Blood-brain barrier (BBB) function can be impaired by free radicals. Menadione can cross the lipid bilayer and is therefore considered as an intracellular. O
2 oxyradical producing agent. It has been shown to increase short term BBB permeability in an in vitro coculture model of immortalized rat brain capillary endothelial and glioma cells. We examined whether exposure of a monolayer of cultured brain endothelial cells to menadione as a generator of intracellular oxidative stress increases BBB permeability in vitro. Materials and methods: The immortalized endothelial cell line bEnd5 from mouse brain capillaries was grown to confluence for 2 weeks in DMEM on semipermeable filters precoated with rat tail collagen. Functional BBB tightness was verified before the permeability assay by measurements of electrical resistance. Culture media was replaced with Ringer-HEPES solution, and radioactive [14C]-sucrose with [3H]-inulin was added to the upper compartment, before filters were successively transferred to other wells containing Ringer-HEPES solution at various time points. Radioactivity was measured in aliquots from the upper and lower compartments, and results

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were calculated as permeability over time elapsed. The endothelial monolayers were exposed to concentrations of 100 lM menadione for 30 min before the permeability assay. In addition, the endothelial cells were exposed to 100 lM menadione during the assay period. Permeability was compared to unstimulated controls, respectively. Results: After exposure to 100 lM menadione for 30 min, the bEnd5 cells showed the same permeability as the controls that had not been preincubated with menadione. When the endothelial cells were incubated directly with 100 lM menadione during the assay, permeability even decreased for the first 6 min as compared to unstimulated controls before it returned to the control level. Conclusion: The results show that unlike in a rat coculture model, incubation of the mouse brain endothelial cell line bEnd5 with menadione, which produces intracellular free radicals, does not increase monolayer permeability. This is contrary to H2O2 as a generator of extracellular free radicals, which has been shown previously to raise monolayer permeability in bEnd5 cells. Therefore, intra- and extracellular free radicals may have different effects on the permeability of brain endothelial cell monolayers in vitro.

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The Endothelium as a Target of Therapy Saturday, July 19, 2003, 16.15
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P49 Immunoliposomal targeting of endothelial cells
therapeutical potential S. Kessner1, U. Rothe2 & G. Bendas1,3 1

Department of Pharmacy, Martin Luther University Halle, Wolfgang Langenbeck Str 4, 06120 Halle, Germany; Medical Faculty, Department of Physiological Chemistry, Martin Luther University Halle, Hollystr. 1, 06097 Halle, Germany; 3Department of Pharmaceutical Chemistry, Rheinische Friedrich Wilhelms University Bonn, An der Immenburg 4, 53121 Bonn, Germany (E-mail: [email protected]) 2

Background: Endothelial cells play an active role in various diseases such as inflammation. They undergo phenotypic modulations to an activated state which is especially marked by expression of several cell surface adhesion molecules. These adhesion receptors are attractive targets for cell selective pharmacological intervention employing drug targeting strategies since they are easily accessible due to the direct contact with the blood. The present study focuses on targeting of E-Selectin by liposomes coupled with selectin antibodies as homing devices. Materials and methods: Freshly isolated HUVEC (IL-1 stimulated) were used as target cells. Liposomes (diameters about 100 nm) were coupled with anti E-Selectin mAb as described, cell binding and internalization under in vitro-conditions were followed by several fluorescence spectroscopical and microscopical means. Results: We could demonstrate that immunoliposomes (IL) bearing anti-E-Selectin antibodies specifically accumulate at activated HUVEC in vitro under both static and simulated blood shear force conditions. In order to derive therapeutical strategies from liposomal targeting, we analyzed route and degree of cellular uptake of the targeted liposomes. Results display that about 25% of targeted IL were internalized by active as well as passive mechanisms. The internalization was correlated with several liposomal parameters. Following the intracellular trafficking of liposomes, we modified the liposomal lipid composition in order to avoid lysosomal degradation. We established pH-sensitive IL that deliver their content into the cytoplasm due to liposome destabilization in the late endosomes followed by liposome-endosome fusion. Consequently, this is a suitable vector system for gene expression. First transfection data were introduced. Conclusion: Selectin-targeted liposomes offer a great potential for therapeutical approaches in the treatment of inflammation, such as liposomal gene therapy.

P50 A functional screen for novel secreted factors that regulate endothelial cell growth and survival S. Hess, T. Walter, R. Scha¨fer, K. Duevell, M. Kazinski, K. Koenig-Hoffmann, I. Boche, A. Bonin-Debs, U. Brinkmann & H. Gille Xantos Biomedicine AG, Max-Lebsche-Platz 31, 81377 Munich, Germany (E-mail: [email protected]) Secreted proteins and their receptors have obvious potential as biological drugs or as targets for humanized antibodies. Secreted growth factors and the interactions with their cognate receptors also represent the highest level of specificity within a given signal transduction pathway as many downstream transducers are often shared between different pathways. In the past, the search for secreted proteins has been driven primarily by computational biology and expression screening efforts. Such approaches have often been restricted to pool transfections resulting in limited sensitivity. Single cDNA clone transfections offer clear advantages with respect

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to sensitivity and hits ought to represent genuine secreted factors whereas false positives and false negatives are generated by signal sequence prediction algorithms. Novel endothelial growth factors are still being identified and there are reasons to believe that other endothelial factors remain to be identified. Such factors can be more easily identified in single clone transfections. Xantos has set up a high-throughput expression screen where cDNA clones are prepared on a robotics platform. We will use our 17,000 clone collection as well as full-length enriched libraries from placenta and tumor tissue. Single cDNA clones are transfected into producer cells using a second robot assembly. Supernatants will be transferred onto two different endothelial cells, human umbilical vein cells (HUVEC) and human microvascular endothelial cells (HMVEC), which are assayed for proliferation. So far we have sreened all 17,000 cDNAs from our clone collection. In addition to known genes including bFGF, VEGF and PDGF-D, we have also found some unknown cDNAs, that have a proliferative effect. Concerning the full-length enriched libraries, we have also screened 22,000 clones from a placenta cDNA library. Currently we are screening 120,000 cDNA clones from tumor tissues. Preliminary results regarding the proliferative factors will be presented. Identified factors may have therapeutic potential in the treatment of diseases where an alteration in the angiogenic activity is considered beneficial such as tumor angiogenesis, diabetic retinopathy or wound healing.

P51 Enhanced monocyte
endothelial cell adhesion by antiretroviral drug treatment S. Hillebrand1, Ch. Huber2, F.D. Goebel1 & B. Banas1 1

Department of Infectious Diseases, Medical Policlinic; 2Department of Virology, Max von Pettenkofer-Institut, University of Munich, Germany (E-mail: [email protected])

Background: HAART has improved the life expectancy of HIV-patients, but is associated with greater cardiovascular risk. This is related to HAART-induced dyslipidemia and glucose intolerance, but eventually also due to direct effects of the drugs on the vascular system. As interactions of endothelial cells (EC) with circulating mononuclear cells (MO) play a crucial role in the development and progression of atherosclerotic lesions, we investigated MO adhesion in respect to antiretroviral drugs. Material and methods: Adhesion Assays (AA) with EC (EA.hy 926) and MO of HIV-patients with different therapy regimen were performed under static conditions. EC were incubated with plain medium or medium supplemented with protease inhibitor (PI-Indinavir) for 24 h. The viral load of the patients was determined by bDNA measurement. Results: The MO-adhesion of HIV infected patients is increased (~60%) as compared to healthy controls. The cell adhesion of therapy naive HIV infected individuals correlates with their viral load, whereas the adhesion of HAART treated patient-MO is increased viral load-independently. Additional incubation of EC with a PI resulted in a synergistic significant increase of MO adhesion to EC (up to 90%). Conclusion: MO of HIV infected patients show an increased MO-EC adhesion in AA. HIV per se changes the adhesive potential of MO, with a positive correlation of high viral load with high adhesion. In patients on HAART MO-EC adhesion is increased independently of viral load (high or low) and PI-regimen (one to three PIs). These experiments indicate that HAART might be associated with additional cardiovascular risk factors that are not yet fully understood.

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P52 Combined retroinfusion of GSH and cariporide attenuates myocardial reperfusion injury in pigs Rabea Hinkel1, Christian Kupatt1, Jan Horstkotte1, Manfred Bilzer2 & Peter Boekstegers1 1

Internal Medizin I, 2Internal Medizin II, Klinikum Großhadern, Munich, Germany (E-mail: [email protected])

Backround: Reperfusion after ischemia may contribute to loss of myocardial function and increase in infarct size. Scavenging of reactive oxygen species (ROS) by Glutathione (GSH) and inhibition of the sodium-protonexchanger by Cariporide are both capable of reducing myocardial reperfusion injury. We tested the efficacy of both agents applied regionally into the ischemia myocardium during reopening the occluded vessel. Methods: Neonatal rat myocytes were used to investigate GSH-accumulation after 1 h of GSH-application under normoxic, hypoxic and reoxygenation conditions. In pigs (n ¼ 5 per group), percutaneous LAD occlusion was performed for 60 min. Application of GSH (250 mg/kg) and/or Cariporide (1 mg/kg) was achieved by pressure regulated retroinfusion through the anterior interventricular vein (AIV), the vessel draining the ischemic area. Retroinfusion was begun 5 min before reopening of the LAD and continued for 10 min. 7 days later subendocardial segment shortening was analyzed by sonomicrometry performed at rest and atrial pacing (120 and 150/min). Infarct size relative to the AAR was determined by Methylen-blue exclusion/Tetrazolium red viability staining. Results: In normoxic and hypoxic cell culture, GSH-content increased by >20-fold after GSH-application for 1 h. Reoxygenation, however, almost completely depleted the cells of the exogenous GSH. In pigs, infarct size of untreated hearts (51%  6%) was unaltered by GSH or Cariporide retroinfusion alone (44%  4% and 42%  6%). Retroinfusion of Cariporide and GSH significantly reduced infarct size (29%  3%). Regional segmental shortening of the infarcted area was improved after Cariporide/GSH retroinfusion as compared to untreated hearts. Conclusion: We conclude that timely and regional application of GSH scavenging ROS and Cariporide targeting ion imbalance is superior to either treatment alone.

P53 Endothelial P-selectin expression as a target of heparin action in experimental melanoma lung metastasis Ralf J. Ludwig1, Christa Tandi1, Jeanette E. Schultz1, Maurizio Podda1, Thomas M. Zollner2, Wolf-Henning Boehncke1, Roland Kaufmann1 & Jens Gille1,3 1

Department of Dermatology, Klinikum der J.W. Goethe-Universita¨t, D-60590 Frankfurt am Main, Germany; RBA Dermatology, Schering AG, D-13342 Berlin, Germany; 3Department of Molecular Biology, Max-PlanckInstitut fu¨r Physiologische und Klinische Forschung, D-61231 Bad Nauheim, Germany (E-mail: [email protected])

2

Spontaneous and experimental metastasis can be effectively inhibited by the widely used anti-coagulant heparin in different tumor models. At the cellular level, many of the anti-metastatic effects by heparin in vivo reflect its action on P-selectin-mediated binding. Whereas previous attention has focused on P-selectin-dependent tumor cell-platelet interactions in blood-borne metastasis, we sought to address the potential contribution of endothelial P-selectin expression to adhesive events between the microvasculature and melanoma cells in vivo. Transplantation of bone marrow from P-selectin-deficient into wild-type mice is shown to convey inhibition of experimental melanoma metastasis. However, the extent to which bone marrow-conferred lack of platelet Pselectin expression attenuated melanoma lung metastasis was significantly less than that seen in P-selectin-

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deficient mice, suggesting that endothelial P-selectin expression may additionally contribute to formation of hematogenous metastases. This assumption is supported by our intravital microscopy studies, detecting a significant proportion of melanoma cells that are capable of directly interacting with postcapillary venules of the murine ear in a P-selectin-dependent manner. Not only does heparin inhibit P-selectin-mediated melanoma cell rolling, but it also attenuates melanoma metastasis formation in vivo, further supporting the concept that endothelial P-selectin expression may represent an additional target of heparin action in experimental melanoma lung metastasis.

P54 Strong antitumor efficacy of a cationic liposomal camptothecin formulation (LipoCam) in the subcutaneous human melanoma A-375 in nude mice H. Haas, B. Schulze, A. Werner, U. Michaelis, B. Sauer & M. Teifel Munich Biotech AG, Forstenriederstr. 10, 82061 Neuried, Germany (E-mail: [email protected]) Background: Cationic liposomes have been shown to preferentially accumulate in angiogenic endothelial cells of pancreatic tumors in transgenic RIP-Tag2 mice (Thurston et al., 1998) and in tumor blood vessels of the amelanotic hamster melanoma A-Mel3 (Krasnici et al., 2003). Recently, cationic liposomes encapsulating paclitaxel (LipoPacTM) have been shown to exert strong antitumor efficacy in several tumor models (Kunstfeld et al., 2003; Schmitt-Sody et al., 2003) and to reduce the mitotic rate of endothelial cells in the tumor periphery (Kunstfeld et al., 2003). Camptothecin (CPT) is known as a potent topoisomerase I inhibitor. Here, we used cationic liposomes to encapsulate CPT (LipoCam) and to target the drug to the tumor blood vessels. In histological sections rhodamine-labeled LipoCam strongly accumulated in tumor blood vessels. Using LipoCam to treat human A375 melanoma in nude mice we observed striking antitumor efficacy resulting in total tumor regression in individual animals at well tolerated doses. Materials and methods: For histological evaluation of the tumor targeting in animals we used rhodaminelabeled LipoCam and analyzed the distribution in different tumors. Therapeutic experiments were carried out using human A-375 melanoma growing subcutaneously into NMRI nu/nu mice. Tumor bearing mice were treated with LipoCam. The animals received nine injections (iv) over a 3-weeks period. Therapeutic effects and tolerability were assessed by analysis of body weight, hematology and tumor volume. Results: Using cationic liposomes, we have developed a cationic lipid/CPT formulation (LipoCam) for (iv) application. LipoCam has an overall positive charge and contains a high amount of CPT. In therapeutic experiments, LipoCam treatment strongly reduced the growth of A-375 melanoma at well tolerated doses. Under the evaluated formulations LipoCam IV showed the strongest antitumor efficacy, resulting in total tumor regression in individual animals. All formulations were well tolerated by the animals. Pathological gross examination revealed absence of macroscopically detectable organ toxicities. Conclusion: (1) Cationic liposomes are a useful tool to deliver CPT to angiogenic endothelial cells. (2) LipoCam showed strong antitumor efficacy in human A-375 melanoma in nude mice.

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The Endothelium in Infectious Diseases Sunday, July 20, 2003, 09.00
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P55 The role of RhoGTPase effectors in the regulation of adhesion and migration of endothelial cells Anna-Eleonor Osiak, Martin Aepfel Bacher & Stefan Linder Institut fu¨r Prophylaxe und Epidemiologie der Kreislaufkrankheiten (IPEK), LMU Mu¨nchen, Germany (E-mail: [email protected]) The integrity and regulation of the endothelial cell layer of blood vessels is of great importance for physiological and pathological processes such as wound healing, growth or inflammation. Endothelial regulation demands an exact coordination of cell movement as well as the dissolution and re-establishing of cell
cell-contacts. The basis for these phenomena is the dynamic and exaxtly controlled regulation of the actin-cytoskeleton. We use a wound healing assay in combination with microinjection, immunofluorescence and live cell imaging techniques to study adhesion and migration of HUVEC. We could observe previously not described actin-rich, rosette-like adhesion structures in HUVEC. They are reminiscent of podosomal adhesions due to v-src transformation of fibroblasts and appear to be typical for migratory HUVEC. We were able to identify N-WASP, Arp2/3 complex, vinculin, c-Src and phosphotyrosine as components of these rosettes. Treatment with cytotoxic necrotizing factor (CNF) of E. coli, an activator of RhoGTPases, greatly reduced rosette formation, indicating the involvement of RhoGTPases in the regulation of these structures. Formation of adhesive actin rosettes in HUVEC is closely linked to a migratory phenotype of these cells. Therefore, rosettes are probably involved in the regulation of endothelial integrity during vessel wall formation, following wounding and after stimulation with bacterial toxins. Future aims include elucidation of the mechanisms involved in formation and regulation of these structures using live cell imaging and microinjection.

P56 High throughput analysis of HHV-8 gene activity in the regulation of cell cycle and apoptosis Frank S. Pogoda, Rene´ Leubert & Michael Stu¨rzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany (E-mail: [email protected]) Background: Human herpesvirus-8 (HHV-8) is highly associated with the pathogenesis of Kaposi’s sarcoma (KS). Progressing KS forms are characterized by increased loads of HHV-8, increased proliferation and decreased apoptosis in the endothelial tumor cells. However, the mechanisms by which the 90 HHV-8-encoded open reading frames may interfere with cell signaling pathways to promote tumorigenesis have not been investigated systematically. To study the influence of HHV-8 genes on cell cycle and apoptosis we use a transfection array to analyze the activation of each HHV-8 gene alone and in combinations. Materials and methods: For transfection array different expression plasmids, alone or in combination are arrayed on slides. The slides are treated with transfection reagent and overlayed with 293T cells. After formation of a confluent monolayer a distinct pattern of cells expressing different viral genes can be analyzed by suitable assays for proliferation or apoptosis.

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Results: We have established and optimized a protocol for a high troughput screening system for gene function based on a transfection array. We printed 0.2 lg/ll expression plasmid DNA diluted in a 0.2% gelatin solution onto slides using an automated arrayer. On this array we achieved a transfection efficiency of 30
50% in 293T cells, as determined by reporter plasmid EGFP-expression. So it is possible to analyze approximately 30
50 transfected cells out of 100 per spot. We have started to establish assays which are applicable to quantitatively determine apoptosis and proliferation of cells in the transfection array. Summary: With transfection array technology it is possible to investigate systematically the function not only of single genes but also of combinations of multiple genes directly in cells. Analysis of combinatory activity of different HHV-8 genes will provide novel insights into pathogenic effect of this virus and its role in KS development.

P57 CD46- and CD150-independent endothelial cell infection with wild-type measles viruses Oliver Andres1, Karola Obojes1, Kwang Sik Kim2, Volker ter Meulen1 & Ju¨rgen Schneider-Schaulies1 1

Institut fu¨r Virologie und Immunbiologie, 97078 Wu¨rzburg, Germany; 2Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA (E-mail: [email protected]) Measles virus (MV) infects endothelial cells (EC) of the skin, the brain and other organs during acute or persistent infections. EC are supposed to play an important role for virus spread from the blood stream to surrounding tissues. CD46 and CD150 (signaling lymphocytic activation molecule, SLAM) have been described as cellular receptors for certain MV-strains. We found that human umbilical vein and brain microvascular endothelial cells (HUVEC and HBMEC) were CD46 positive, but did not express SLAM. In addition, treatment of the cells with LPS, IL-1beta, TNF-alpha, or MV did not induce the expression of SLAM. In spite of the absence of SLAM, wildtype MV-strains, which use SLAM but not CD46 as receptor at the surface of transfected CHO cells, infected HUVEC and HBMEC in extents varying in a strain dependent way. The infection was not inhibited by antibodies to CD46. These data suggest the presence of an unidentified additional receptor for MV uptake and spread on human endothelial cells.

P58 Impact of hydrocortisone on measles virus replication and viral induction of the immune response in endothelial cells Karola Obojes1, Stefanie Nicklaß1, Oliver Andres1, Kwang Sik Kim2 & Ju¨rgen Schneider-Schaulies1 1

Institut fu¨r Virologie und Immunbiologie, 97078 Wu¨rzburg, Germany; 2Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA (E-mail: [email protected]) Measles virus (MV) infects endothelial cells (EC) of the skin, the brain and other organs during acute or persistent infections. The extent of EC infection during acute measles varies broadly and can develop clinical pictures from limited petechia of the typical exanthema up to massive haemorrhagic lesions. Factors influencing the extent of MV replication in EC in vivo are not known. Hydrocortisone (HC) is now widely used in EC cultures to stimulate barrier properties. However, when EC interactions with infectious agents are investigated, the immunosuppressive effects of HC must be taken into account. We found that MV replicates considerably better in HC treated human umbilical vein and brain microvascular endothelial cells (HUVEC and HBMEC) than in untreated cells. It is known that HC suppresses the induction of type I interferon and other cytokines which constitute the inflammatory response of cells to a viral infection. The effects of HC on MV replication, the induction of cytokines by MV-infection, and transmigration of MV-infected leukocytes are currently investigated.

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P59 The role of the staphylococcus aureus extracellular adhesive protein (Eap) on the interaction with endothelial cells Astrid Sobke1, Muzaffar Hussain2, Bhanu Sinha2, Triantafyllos Chavakis4, Klaus T. Preissner3 & Mathias Herrmann1 1

Department of Bacteriol. and Hygiene, University of Saarland, Homburg/Saar, Germany; 2Inst. of Med. Microbiol., University of Muenster, Germany; 3Department of Biochemistry, University of Giessen, Germany; 4 Department of Medicine, University of Heidelberg, Germany (E-mail: [email protected]) Background: Staphylococcus aureus is a major pathogen causing endovascular diseases such as infective endocarditis, mycotic aneurysms, or suppurative thrombophlebitis. Traditionally being regarded as an extracellular pathogen, it is now clear that S. aureus invades endothelial cells, which may be a prerequisite for endovascular disease. Invasion is dependent on a functional fibronectin-binding protein (FnBP)-fibronectina5b1 integrin bridging [Sinha et al. Cell Microbiol 1999; 1: 101). However, the major secreted adhesin Eap may partially compensate for loss of FnBP-mediated invasion (as shown for S. aureus Newman; Haggar et al., IAI 2003, in press) and modulates immune functions through ICAM-1 dependent mechanisms (Chavakis et al., Nature Med 2002; 8: 687). The goal of this study was to investigate effects of Eap on endothelial cell invasion and intracellular signaling. Materials and methods: Confluent mouse microvascular endothelial cells (bEND) were exposed to Eap (extracted from S. aureus Newman by LiCl treatment followed by cationic exchange chromatography) and/or purified staphylococcal peptidoglycan (PG). Invasion was quantified by a lysostaphin protection assay. Whole cell lysates were subsequently analyzed by Western blotting with phospho-specific antibodies against JNK, ERK1/2 or p38. Results: Eap binding to bEND cells was confirmed in Western blots and by immunocytochemistry. Adhesion and invasion of S. aureus Newman WT appeared enhanced as compared to a Deap mutant. Use of different Eap preparative methods suggests that contamination with (PG) may contribute to intracellular effects. First assays revealed that purified Eap (up to 100 lg/ml) induced ERK1/2 and p38 kinase phosphorylation. Conclusions: Eap interacts with endothelial ligands such as ICAM-1, and has multi-faceted effects on the interaction of S. aureus with the endothelium. In addition to adhesion and invasion, Eap appears to modulate intracellular signalling through pathways involving the MAP kinase family. Further analysis will determine mechanisms and consequences of this interaction in detail.

P60 Cationic liposomes for tumor imaging: First clinical results Ruth Knuechel1, Kerstin Bartelheim2, Oliver Reich3, Ralf Oberneder3, Herbert Stepp3, Uwe Michaelis2, Kurt Naujoks2 & Birgitta Sauer2 1

Institute for Pathology, University of Regensburg, Regensburg, Germany; 2Munich Biotech AG, Neuried, Germany; 3Department of Urology and Laser Research Laboratory, Ludwig Maximilians University Munich, Munich, Germany (E-mail: [email protected]) Background: A tumor selective delivery of agents is the topic of many research efforts in oncology. Cationic liposomes have been shown to preferentially accumulate in angiogenic endothelial cells of tumor vessels in pancreatic tumors and melanoma (Thurston et al.; 1998; Krasnici et al., 2003). Here, we investigated the neovascular targeting of Rhodamine (rh) labeled cationic liposomes (LipoRedTM) developed for tumor imaging in tumor biopsies derived from patients of a clinical Ib/II trial in bladder cancer. First clinical data clearly demonstrated that LipoRedTM fluorescence is found to be increased in tumor lesions macroscopically and microscopically specifically attached to tumor blood vessels.

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Materials and methods: LipoRedTM was administered by iv. infusion to patients at different doses. The tumor specific accumulation of LipoRedTM was evaluated intrasurgically by fluorescence spectrometry and verified by histopathology of tumor and tissue samples. Images were taken with a high resolution camera and digitally processed. Results: In a clinical Ib/II trial in bladder cancer tumor lesions clearly exhibit an increase of liposomal fluorescence after iv. application of the imaging agent LipoRedTM evaluated by intrasurgical spectrometry. Histological analysis of tumor sections revealed that liposome related fluorescence was limited to capillaries and medium sized vessels. Interestingly, stromal capillaries in areas of carcinoma in situ and pT1-carcinomas showed highest fluorescence in proximity to tumor cells indicating association to neo-angiogenesis. Capillaries in inflammatory stroma did show liposome binding, however less fluorescence. Normal mucosa and deeper areas of the bladder wall with larger vessels was devoid of rh fluorescence. The administration of the new drug proved to be safe at the current dose levels. Conclusion: (1) After systemic application of LipoRedTM in humans a striking endothelial specificity preferentially for neo-vascular tumor blood vessels was demonstrated. (2) The finding of a preferential uptake of LipoRedTM correlating with precancerous and invasive stages of tumors may be used for the delivery of therapeutic drugs encapsulated in cationic liposomes.

L61 The Thy1/Thy-1-ligand-interaction is involved in binding of peripheral blood cells as well as melanoma cells to activated Thy-1-positive microvascular endothelial cells A. Saalbach, A. Wetzel, U.F. Haustein, M. Sticherling & U. Anderegg Department of Dermatology, University of Leipzig, Germany (E-mail: [email protected]) Cell adhesion plays a central role in inflammation as well as in metastasis of tumor cells. Cell adhesion mediated by specific adhesion molecules direct and regulate the invasion of cells in certain tissues. In recent studies, we characterized the human Thy-1 molecule as an inducible activation associated cell adhesion molecule on human dermal microvascular endothelial cells (HDMEC). Thy-1 expression on microvascular endothelial cells can be induced in vitro by TNF-alpha and Phorbolesters and is found in vivo at sites of inflammation and tumor growth. Thy-1 has never been detected on resting endothelial cells in situ and in vitro. Our experiments proved the involvement of the Thy-1/Thy-1-ligand interaction in binding of polymorph nuclear cells and monocytes to activated Thy-1-positive HDMEC. The Thy-1-ligand was detected on polymorph nuclear cells and monocytes. Protein binding and cell adhesion studies demonstrated that CD11b/CD18 is a natural ligand of Thy-1 on circulating blood cells. The strength of this interaction was comparable to the interaction of ICAM-1 with this integrin. These data suggest a specific and powerful role of Thy-1 during binding of monocytes and polymorph nuclear cells to activated endothelium. Furthermore, we could demonstrate a strong expression of Thy-1 on endothelial cells in tissue sections of malignant melanoma. In vitro, an induction of Thy-1 expression on HDMEC was found after stimulation with melanoma cell derived soluble mediators. The Thy-1-ligand was found on different melanoma cell lines in vitro and on melanoma cells in situ. We could demonstrate, that this so far unknown ligand is different from the CD11b integrin shown for blood cells. In cell adhesion assays the involvement of the Thy-1/Thy-1-ligand interaction in binding of melanoma cells to activated Thy-1-positive HDMEC was shown. Taken together, the investigation of Thy-1 and Thy-1 ligand expression might allow a more detailed insight in the direction and regulation of the invasion of cells in tissues with inflammation as well as during metastasis of certain tumor cells.

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P62 Thy-1 (CD90) determines a subpopulation of activated human dermal microvascular endothelial cells (HDMEC) exhibiting an expression pattern of lymphatic endothelium in vitro Anja Saalbach, Knut Krohn, Anne Wetzel, Michael Sticherling & Ulf Anderegg Department of Dermatology, Leipzig University and Saxon Academy of Science, Leipzig, IZKF Leipzig, Germany (E-mail: [email protected]) Microvascular EC represent a heterogeneous population differing along blood and lymph vessels, in various organs and fulfilling various functions. Reliable cell markers are the major prerequisite for the detection and analysis of those subpopulations. We have demonstrated that human Thy-1 is an activation marker on a subpopulation of HDMEC. These subpopulations can be separated using antibody coupled microbeads and analysed towards cell biological functions and expression patterns. Microarray analysis was performed to identify differentially expressed genes in Thy-1 (+) vs. Thy-1 ()) cells. Two RNA pools of Thy-1(+) and Thy-1()) activated HDMEC each consisting of five independent separations were used for four hybridisations of Affymetrix U95 chips. The hybridisation intensity data were compared from all pools generated among each other (four comparative analyses). About 100 genes showed expression differences of >2-fold after each comparison of Thy-1(+) vs. Thy-1()) cells. The data were strongly reproducible in Realtime-RT-PCR analysis for highly differently expressed genes (32-fold increase) as well as for weakly increased genes (2-fold increase) proving the reliability of the data from the microarray experiment. In vitro Thy-1 expression is induced on a subpopulation of human dermal microvascular EC after activation with phorbolester, TNF-alpha or melanoma cell derived factors resulting in two cell populations distinguished by their Thy-1 expression (Thy-1-(+) and Thy-1-()) EC). Gene expression profiling showed, that Thy-1-(+) EC display an expression pattern being very similar to lymphatic EC. Furthermore, Thy-1-()) EC express genes as described for BEC. Thy-1 is a marker of a subpopulation of activated BEC and LEC, those gene expression pattern is converted to a LEC-like phenotype. Our results underline the observation that differentiated EC might be able to reprogram their gene expression pattern.