The cardiovascular system is vital for nutrient delivery and blood pressure maintenance throughout the body. Although approximately 1.2 million Australian adults have conditions related to heart or vascular disease, we are still understanding the complex cellular signalling pathways that regulate blood vessel growth and stability. Blood vessels must be able to sense and react to a range of environmental cues to maintain their structure and signalling, and to generate new vessels when the need arises. An important aspect of engaging with these cues is the ability of the endothelial cells (that make up the inner lining of blood vessels) to engage in crosstalk with the extracellular matrix (ECM). Although it is established that endothelial cells have the ability to translate mechanical signals into biochemical signals, there is a gap in our knowledge on how this occurs, and how it is dysregulated in disease.
We recently identified the non-receptor tyrosine kinase, c-Src, as a driver of blood vessel growth and cell adhesion signalling. In conditional, endothelial cell-specific knock out mice, loss of c-Src significantly reduced blood vessel sprouting and focal adhesion signalling within the vessels. To further investigate specifically how c-Src regulates these pathways, we have generated a range of lentiviral constructs which express fluorescently tagged c-Src mutants. These plasmids express wild-type (WT), constitutively active (Y527F, CA), or dominant negative (Y527F-K295R, DN) c-Src. Cultured endothelial cells were transduced with these plasmids to study the effect of c-Src activity in a 3D fibrin bead sprouting assay and microfluidic vessels. In both 3D models, CA-c-Src induced balloon-like vascular malformations with large gaps between the endothelial cells, indicating loss of vessel integrity. CA-c-Src also caused a decrease in fibronectin remodelling and localised degradation of ECM specifically under large focal adhesions. As matrix metalloproteinases (MMPs) are known to be secreted at focal adhesions, we treated cells with a broad-spectrum inhibitor, Marimastat, which rescued the effects of CA-c-Src. This suggests that increased c-Src activity drives blood vessel defects by increasing matrix degradation at focal adhesions. As the process of focal adhesion dynamics is not yet fully understood, these findings contribute another key piece to the puzzle of the role of focal adhesions in endothelial cell biology.
Overall, we hope to identify new targets in diseases where vascular growth and integrity contributes to disease burden, such as the tumour microenvironment.