High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers

Endothelial cells (EC) are heterogeneous both across different tissues and within the same tissue, reflecting distinct specialised microenvironments and functions. Microenvironment independent EC heterogeneity within the same vascular bed has been appreciated and proposed to underpin EC monolayers plasticity. Nonetheless, the underlying molecular mechanisms are elusive. Heterogeneity within the same monolayer driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with gold standard single cell transcriptomic approaches as they don9t allow extracting spatial and context information. Dissecting the molecular mechanisms leading to EC heterogeneity is key to offers novel therapeutic targets for cancer and cardiovascular diseases and for revascularisation strategies in regenerative medicine. Here we developed an EC profiling tool (ECPT) to enable profiling of individual EC within a monolayer providing spatial and relational information on cell behaviour (including cell proliferation, inter-endothelial Junctions and NOTCH activation). We used ECPT to characterise differential phenotypes in arterial (Aortic EC), venous (HUVEC) and microvascular (Pulmonary microvascular EC) EC populations. We find that different EC populations display a remarkable heterogeneity in terms of cell cycle and proliferation, cytoskeleton organisation, junction stability and a previously under-appreciated single-cell NOTCH heterogeneity. We also used ECPT to correlate junction stability with different NOTCH activation states at the single cell and population levels. In conclusion ECPT is an extremely versatile tool to study positional and relational information in EC monolayers paving the way towards pharmacodynamic screenings and synthetic biology applications and overall, a better understanding of EC biology.