P305	Integrin Linked Kinase (ILK) plays an important role in embryonic vasculo- and angiogenesis.
1A.Elischer, 1A.Schmidt, 2B.Fleischmann, 1W.Bloch, 2D.Malan
1German Sport University Cologne, Cologne, DE; 2Institute of Physiology I University of Bonn, Bonn, DE.

The extracellular matrix (ECM) is essential for modulating processes such as proliferation, apoptosis, migration and differentiation of cells. This regulation is largely dependent, among other extracellular matrix proteins, on integrins, a family of transmembrane-receptors, which activate intracellular signaling molecules by interacting with various effector proteins. Integrin-linked kinase (ILK), a serin/threonin kinase, is a key molecule of the cell-ECM adhesion. It interacts with the actin cytoskeleton and with the β1-integrin cytoplasmic domain. Since recent studies have shown the importance of the integrity of β1-integrins and consequently of the cytoskeleton organization in receptor signalling and clustering as well as in vasculogenesis, we analysed the role of ILK in these processes. As model of embryonic vasculo-and angiogenesis, EBs (embryoid bodies) from mouse embryonic wild type ES line and ILK deficient (-/-), at different stage of development (5+7 and 5+14), were generated. ILK deficient EBs showed a disrupted morphology of endothelial tubes as well as a decreased number of vessel-like structures. The analysis of ECM by immunocytochemistry experiments revealed major defects in collagen IV, laminin and fibronectin deposition, whereas perlecan, which does not bind to β1-integrins, was intact. Additionally, proliferation and apoptosis-experiments were performed by analysis of the number of Ki67 and Caspase-3-positive nuclei per 25 tube-like structures, respectively. In both cases a significant higher rate of the knockout- compared to the wild type-cells was observed. We further analysed the involvement of ILK on VEGF signalling pathway by [Ca2+]i imaging experiments on magnet-associated cell sorting (MACS)-sorted endothelial cells from wild type and ILK (-/-) EBs loaded with the dye fura-2. Wild type cells after perfusion with VEGF (20ng/ml) displayed a classical [Ca2+]i transient which was absent in most of the ILK (-/-) cells. So far, these experiments point out the important role of ILK in embryonic vascular development by affecting ECM stability, proliferation, apoptosis and by regulating morphogenesis as well as signalling pathway.

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