Introduction: 

During vascular remodelling proteases are activated and show significant modulator properties for cellular adhesion leading to adhesion dependent apoptosis (anoikis). Within the extracellular matrix, laminin belongs to the most abundant non-collagenous proteins and exhibits important regulatory potential for the vascular development. Therefore we were interested in the apoptotic signalling on endothelial cells (EC) affected by laminin degradation. Elastase digests laminin into distinct parts of which the fragment E8 is bound by integrins. In contrast the pepsin fragment P1 is bound solely by the anti-apoptotic laminin binding protein LBP. Using purified fragments we sought to dissect the cellular signalling caused by either of these receptors.

Methods and Results: 

Seeding EC on purified E8 resulted in a reduced proliferation (p<0.05, n=8), quantified with MTT using FGF-2 as stimulus. EC grown on E8 exhibited a 2-fold higher apoptosis rate (annexin V translocation n=4, p<0.05) as well as increased reactive oxygen radical (ROS) production (by DCF, 1.10.5nmol/min vs. 2.31nmol/min). Aortic ring sprouting was significantly inhibited by E8 and was revoked by both radical scavengers SOD and catalase. In contrast to the findings with the laminin fragment E8 the fragment P1 showed characteristics not significantly different from intact laminin. Proliferation, apoptosis and ROS production were unchanged indicating that LBP, which exclusively binds the fragment P1, 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 the missing LBP binding to laminin induced apoptosis in EC (2.5-fold over control), similar as for fragment E8. Furthermore co-precipitation experiments showed that LBP interacts with death associated protein 3 (DAP-3) and suggests, consistent with data from the literature, that LBP exhibits beside its properties as matrix receptor a significant role in the regulation of matrix induced apoptosis (anoikis).

Conclusion: 

Due to their binding to laminin, integrins and LBP are under normal condition in close proximity within the same focal adhesion point. In contrast to intact laminin, degradation of laminin to the fragment E8 inhibits proliferation and angiogenesis and induces apoptosis and ROS production. These results indicate 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 feed back loop to limit proliferation and differentiation of EC in adaptive vascular remodelling.