Endothelial Cell Organization Drives Distinct Agonist‐Specific Ca ²⁺ Dynamics in Arteries and Veins

Aim The endothelium regulates cardiovascular function by detecting and interpreting multiple extracellular signals from blood and surrounding tissues, even when these inputs are complex and conflicting. The major challenge faced by the endothelium is decoding this dynamic chemical environment to produce coordinated endothelial cellular responses. In addition to the problems of detection, extracellular signals must be processed correctly intracellularly to generate a functional outcome. Methods Ca 2+ imaging, network analysis and spectral graph theory across ~1000 endothelial cells in intact arteries and veins. Results The venous endothelial cell population forms distinct, non‐overlapping communities, each tuned to specific agonists. Within these communities, responsive cells act as bridges, linking members through the most direct communication route. Activation of one cell increases the likelihood of activation occurring in its neighbors, creating localized zones of high responsiveness. Only a small (5%) subset of cells responds to multiple activators. These multifunctional cells form unique connections that integrate and distribute signals between the agonist‐specific sensing communities. We also show that different agonists elicit unique signaling patterns determined by the stimulus, not by intrinsic cellular properties. Finally, signal decoding strategies differ across vascular beds: venous endothelial cells rely on Ca 2+ signal frequency, while arterial cells use signal amplitude. Conclusion The endothelium comprises functionally specialized populations. A small subset of pharmacologically distinct cells plays a key role in signal integration. These hubs are especially vulnerable to disconnection and dysfunction in disease, highlighting them as potential therapeutic targets. The findings presented reveal specialized encoding strategies that distinguish the arterio–venous axis.