High-content image analysis to study phenotypic heterogeneity in endothelial cell monolayers

Area of Science:

• Vascular Biology
• Cell Biology
• Biotechnology

Background:

• Endothelial cells (ECs) exhibit significant heterogeneity, influencing their specialized functions and plasticity.
• Current single-cell transcriptomic methods lose crucial spatial and contextual information, limiting the study of cell-cell crosstalk.
• Understanding EC heterogeneity is critical for developing therapeutics for cancer, cardiovascular diseases, and regenerative medicine.

Purpose of the Study:

• To develop and utilize a novel tool for analyzing endothelial cell heterogeneity within intact monolayers.
• To characterize distinct endothelial cell phenotypes in arterial, venous, and microvascular populations.
• To investigate the relationship between endothelial cell proliferation and NOTCH activation states at a single-cell level.

Main Methods:

• Development of an Endothelial Cell Profiling Tool (ECPT) for examining individual cells in intact monolayers.
• Application of ECPT to characterize phenotypes in arterial, venous, and microvascular ECs.
• Quantification of heterogeneity in cell cycle, junction organization, and NOTCH activation.

Main Results:

• ECPT successfully characterized endothelial cell heterogeneity, including cell cycle, proliferation, and junction organization.
• A previously unrecognized single-cell heterogeneity in NOTCH activation within endothelial cells was uncovered.
• Cell proliferation was correlated with different NOTCH activation states at both single-cell and population levels.

Conclusions:

• The developed ECPT provides crucial positional and relational information for studying endothelial cell heterogeneity.
• This approach advances the understanding of molecular mechanisms driving endothelial cell heterogeneity and plasticity.
• Findings pave the way for targeted therapeutic strategies in vascular-related diseases and regenerative medicine.