O211	Role of Laminin γ1in vascular development and differentiation.
1D.Malan, 1A.Schmidt, 3N.Smyth, 2B.Fleischmann, 1W.Bloch
1German Sport University Cologne, Cologne, DE; 2Institute of Physiology I University of Bonn, Bonn, DE; 3Institute of Biochemisty University of Cologne, Cologne, DE.

We investigated the role of extracellular matrix in direct vasculo- and angiogenesis in a murine embryonic stem (ES) cells model deficient (-/-) for laminin γ1 chain, one of the most ubiquitous chains of all laminins. Laminin is a heterotrimeric extracellular matrix glycoprotein which can interact with structural molecules of basement membrane and seems to be involved in its deposition and organization. Laminin γ1 deficiency caused changes in the morphology of vessel-like structures in early and late stages (5+7 and 5+14, respectively) embryoid bodies (EBs) compared to the wild type. We observed smaller and disorganized tube-like structures in early stages in the laminin knock out compared to the wild type as well as less number of tube-like structures in late stages for laminin deficient EBs. The assembly of a basement membrane around the tube-like structures failed, as shown by confocal images for collagen IV and perlecan deposition, whereas an increase of fibronectin deposition was observed in the laminin (-/-) vessel-like structures. Moreover, reconstitution experiments with laminin 1 (25ng/ml) pre-treated EBs showed a partial deposition of basement membrane around the tube-like structures of laminin (-/-) EBs indicating a specific effect. Lacking of laminin γ1 determined higher rate of proliferations, increasing Ki67-positive nuclei per 50 tube-like structures in early stages (wild type 21.6 ± 6.0 and (-/-) 104.1 ± 3.7). Proliferation rate was not changed for late stages, moreover apoptosis rate detected as the number of PARP-positive nuclei per 50 vessels was not enhanced in both stages. The migration of endothelial cells was enhanced in late- compared to the early-stage but laminin deficient cells had a smaller rate. By densitometry analysis we observed a up-regulation of Akt and NOS Ser(1177) phosphorylation indicating a disruption in the proliferation signalling pathway. Thus, laminin γ1 role is not only in basement membrane assembly but in the maintenance of stability of developing vascular tube through the modulation of NO pathway and regulation of proliferation.

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