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Bidzhekov, Kiril (2005): Investigations on endothelial maturation and anticoagulant properties. Dissertation, LMU München: Medizinische Fakultät



Mature endothelial cells are terminally differentiated cells with a low proliferative potential and their capacity to substitute damaged endothelium is limited. Accumulating evidence in the last years indicates that mammalian organisms contain a unique subtype of circulating, bone marrow-derived cells with properties similar to those of embryonal angioblasts. These cells were called endothelial progenitor cells (EPCs). In the present work, we have studied the role of B-Raf and C-Raf, two members of a central intracellular signalling pathway, for the proliferation and differentiation of mouse embryonic EPCs. A further purpose of the study was to evaluate the anticoagulant properties of the mature endothelium and in particular the role of Tissue factor pathway inhibitor (TFPI). We prepared gene constructs allowing us to activate or inhibit the downstream signalling of B-Raf and C-Raf, and on the other side we have used RNA interference to knock down these proteins. We found that both B-Raf and C-Raf are engaged in the proliferation of the eEPCs. However, B-Raf is mostly responsible for the differentiation, and cAMP is activating the differentiation through B-Raf, but not through C-Raf. To delineate the participation of the endothelium in coagulation, the role of native TFPI and its mutated forms in intravascular fibrin formation was analyzed. Particular attention was given to TFPI mutants being resistant towards cleavage by leukocyte proteases that might inactivate TFPI under physiological and pathophysiological conditions. The novel insights on the differentiation and proliferation of the endothelial progenitor cells obtained in the present work, may give us the opportunity to regulate their functionality in certain cases, and eventually using them as therapeutic agents in some kind of diseases (e.g. myocardial infarction, stroke).