Investigations on endothelial maturation and anticoagulant properties

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).