Blood vessels run throughout our body to supply oxygen and nutrients to all tissues. To maintain their function, they must adapt to the characteristic stiffness of the different tissues that surround them. Depending on the function, each tissue has a specific stiffness which is defined by their extracellular matrix (ECM), and which differs greatly, e.g. bone is stiff to provide structural support to the body, while lungs are soft to provide flexibility to inflate and deflate during breathing. Endothelial cells that line the blood vessels are highly sensitive to mechanical signals such as ECM stiffness, and the exponential increase of mechano-signalling research reflects its importance. Studying the effect of ECM stiffness on endothelial cells, we found that increased stiffness is corelated with increased activation of the non-receptor tyrosine kinase c-Src and with increased formation of focal adhesions (FAs).
To further investigate the molecular mechanism of how induction of c-Src activity by changes in matrix stiffness regulates endothelial functionality, we generated fluorescently tagged wildtype (WT), constitutively active (CA) and kinase dead (KD) c-Src constructs. In cultured endothelial cells, we found that WT c-Src promoted formation and maturation of FAs shown by increased FA number and size. These changes were further elevated by the CA c-Src mutant and rescued upon expression of the KD c-Src mutant, revealing that activation of c-Src in endothelial cells regulates cell-matrix adhesion through FAs.
We then assessed cell migration as a readout for endothelial cell functionality upon increased c-Src activity. Where expression of WT and KD c-Src mutants showed no difference in migration velocity and distance, CA c-Src reduced migration velocity and distance in a 2D wound healing assay. Finally, c-Src signalling has been implicated in regulating cell-cell adhesion through the junctional protein VE-cadherin, where phosphorylation of VE-cadherin by c-Src stimulates VE-cadherin internalisation and subsequently reduces cell-cell adhesion. Using the c-Src mutants, we found that activation of c-Src increased VE-cadherin phosphorylation and internalisation, resulting in gap formation between the endothelial cells.
In conclusion, our results suggest that increased ECM stiffness activates c-Src in endothelial cells which in turn induces endothelial dysfunction by disrupting cell-cell junctions and enforcing cell-matrix adhesions. Future studies, using novel 3D micro vessels will uncover how changes in ECM stiffness affect endothelial functionality in physiological settings, and whether c-Src may be a good target to prevent vascular dysfunction induced by a stiff ECM.