|Abstract||The Protein Kinase D (PKD) family is set up by three highly conserved members, the isoenzymes PKD1, 2 and 3. PKDs are diacylglyerol-stimulated serine/ threonine protein kinases and form a subgroup of the Calcium/ Calmodulin Protein Kinase (CAMK) superfamily. Various stimuli lead to a Protein Kinase C (PKC)- dependent PKD-activation and the performance of multiple functions in diverse biological systems. A major role in physiological as well as tumour angiogenesis is attributed to PKD2. Angiogenesis is referred to as formation of new blood vessels from pre-existing vascular structures by endothelial cell differentiation. In contrast, vasculogenesis describes de novo blood vessel formation by endothelial cell differentiation from mesoderm during development. PKCs, the prominent upstream activators of PKDs, are known to drive lineage commitment in mouse embryonic stem cells (mESCs). Moreover, in the developing embryo, vasculogenesis and angiogenesis are closely linked. However, the role of PKDs during early embryonic cell fate decision and especially vasculogenesis largely remains elusive.
To address this question, this study uses a combined gain- and loss-of-function approach in mouse embryonic stem cells for in vitro experiments, complemented with chicken chorioallantoic membrane (CAM) xenografts for an in vivo approach. This study demonstrates PKD2 to be strongly expressed in mESCs, followed by PKD3 and PKD1 only presenting marginal expression levels. For the first time, we describe time-restricted PKD2-activity influencing early cell fate decision and angiogenesis. During early differentiation (days 0-4), PKD2-activity decreases mesendoderm formation and subsequent cardiovasculogenesis in favour of ectoderm, while promoting sprouting angiogenesis during late differentiation (days 4-14). Consistently, PKD2-loss-of-function analyses demonstrate opposed effects. These findings are confirmed in vivo, as embryoid bodies (EBs), transplanted on CAMs, induce an angiogenic response upon PKD2-overexpression from day 4 of differentiation onwards.
Summing up, this thesis presents novel and time-dependent aspects of PKD2- activity during early embryonic development. Our data provide further insight into the complex regulation of angiogenesis, that might even be of clinical interest in the fields of cancer research and regenerative medicine.||dc.description.abstract