High Content Analysis uncovers functionally relevant metastable phenotypic states in human Endothelial Cells monolayers

Endothelial cells (EC) present distinct cell properties in different tissues. Heterogeneity within the same vascular bed has been proposed to underpin emergent behaviours in EC monolayers. Quantification and functional relevance of EC phenotypic variance are challenging to assess. Here we developed a novel EC profiling tool (ECPT) to uniquely enable single EC profiling within a monolayer and providing spatial and relational information regarding cell proliferation, inter-endothelial Junctions and NOTCH activation. We used ECPT to characterise differential phenotypes in arterial, venous and microvascular EC populations. Our analysis highlighted extensive heterogeneity within individual monolayers and revealed VEGF-modulable metastability of NOTCH signalling which in turn regulates inter-endothelial junctions` stability. We suggest that accounting adaptive emerging endothelial behaviours is necessary to develop specific tissue engineering approaches and identify more effective and EC-specific therapeutic targets for cardiovascular diseases and cancer.