Introduction: 

Mechanical loads within the vascular wall lead to adaptative remodelling in order to cope with those new demands. Laminin belongs to one of the most abundant non-collagenous proteins within the extracellular matrix and exhibits important regulatory potential for the vascular development. Thus, we were interested in endothelial cells (EC) signalling mechanisms initiated by laminin receptors. Using purified laminin fragments, we sought to dissect the cellular signalling events caused by either of these receptor types and tried to identify the interaction of those diverse matrix receptors. Integrins are primarily binding to fragment E8 and the "laminin binding protein RPSA" (former LBP) recognizes only laminin fragment P1/E3. Based on data from the literature identifying RPSA to be part of an anti-apoptotic cell-program, we hypothesized that fragmentation of laminin might show significant modulator properties for cellular adhesion leading to adhesion dependent apoptosis (anoikis).

Methods and Results: 

Seeding EC on purified E8 resulted in a reduced proliferation, quantified with MTT. Those cells showed further a nearly 2-fold higher apoptosis rate (annexin V translocation) as well as increased reactive oxygen radical (ROS) production (by DCF and cytochrome C assay). Aortic ring sprouting was significantly inhibited by addition of purified E8 to the embedding matrix. Both SOD and/or catalase treatment revoked the inhibitory effect of E8 indicating oxygen radical formation as effective mediator. In contrast to the findings with fragment E8, fragment P1/E3 showed characteristics not significantly different from intact laminin. Proliferation, apoptosis and ROS production were unchanged indicating that RPSA/LBP, which exclusively binds the fragment P1/E3, dependent signalling efficiently inhibited apoptosis. In order to proof this concept LBP binding to intact laminin was interrupted using the peptide YIGSR. As expected, even in case of intact laminin as substratum the lack of RPSA/LBP binding to laminin induced apoptosis (2.5 fold over control), similar as shown for fragment E8.

Conclusion: 

Our data are consistent with the literature, indicating that RPSA/LBP exhibits beside its properties as matrix receptor a significant role in the regulation of matrix induced apoptosis and anoikis. In contrast to intact laminin, degradation of laminin to fragment E8 inhibits proliferation and angiogenesis and induces apoptosis and ROS production. These results imply that proteolytic alteration of the matrix post synthesis induces distinct signalling by changes of the focal arrangement of laminin receptors and might by a mechanism, which could be part of a negative feed back loop to limit proliferation and differentiation of EC in adaptive vascular remodelling.