Introduction: 

Endothelial cells are important components of the cellular vascular system and function as sensor for multiple signals. Among those, mechanical loads like shear stress within the vascular wall are very important leading on the short time period to the synthesis and release of vasoactive substances and on the long run to adaptative remodelling in order to cope with these new demands. During vascular remodelling proteases are recruited and seem to be critically involved in the generation of cellular signalling cascades. In this study we follow the hypothesis that due to the proteolytic activity the elasticity of the extracellular matrix is changed and by that induces cellular responses. We focussed on the matrix component laminin as the proteolytic target because it 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 investigated endothelial cells (EC) signalling mechanisms initiated by laminin degradation. We found earlier that a distinct elastase, which belongs to serine-proteases, is activated/released during shear stress. 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 matrix binding leading to adhesion dependent apoptosis (anoikis).

Methods and results: 

C were seeded on to either laminin or fibronectin coated polyacrylamide gels exhibiting increasing rigidity (stiffness). The estimated Young modulus ranged from 7 to 100 kN/m² and was compared to a plastic substrate which reflects the classical culture condition. In parallel to a decreasing stiffness, EC showed significant changes in their morphology from a flat and spread phenotype to a more rounded and contracted verifying that those cells are constantly pulling on their adhesion sites. Weak and flexible matrix gels gradually induced apoptosis, which was quantified by counting fragmented nuclei. Similarly, EC grown on either laminin or fibronectin and which were exposed to elastase (0.5U/ml) for various time periods (from 15 to 120 min), showed increasing apoptosis rates (tunel assay) which was significantly more pronounced on laminin than on fibronectin. A combination of various matrix stiffness and exposure to elastase showed that notably on a firm (stiff) underground elastase treatment induced an additional increase of apoptosis. Seeding EC on purified laminin fragments, which are bound by distinct integrin and non-integrin receptors resulted in a reduced apoptosis rate (annexin V translocation) as well as increased reactive oxygen radical (ROS) production (by DCF and cytochrome C assay) if the cells were lacking an adhesion via the non-integrin laminin receptor.

Conclusion: 

Due to their binding to laminin, integrin and non-integrin receptors are under normal condition in defined proximity within the same focal adhesion point. In contrast to intact laminin, degradation of laminin 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 matrix receptors due to changes in the matrix flexibility. Furthermore, changes in the mechanical load of the vascular wall in a micro-scale range could represent initiator signalling sites not only for adaptive remodelling but also even for apoptosis. We suggest that the use of gels with distinct rigidity might serve as cell models not only for aneurisms but also for arteriosclerosis.