Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell function and vascular structure. While vascular responses to flow are complex and context-dependent, endothelial cell signaling in response to shear stress induced by laminar flows is coordinated by the transcription factor KLF2. The flow-dependent expression of KLF2 in endothelial cells is associated with a quiescent, anti-inflammatory phenotype and has been well characterized in two-dimensional systems but has not been studied in threedimensional in vitro systems. Here we develop engineered microvascular networks (MVNs) that incorporate a KLF2-based endothelial cell flow sensor within a microfluidic chip, apply continuous flow using an attached microfluidic pump, and study the effects of this flow on vascular structure and function. We found that application of flow to MVNs for 48 h resulted in increased expression of the KLF2 reporter, larger vessel diameters, and decreased vascular branching and resistance. Notably, vessel diameters after the application of flow were independent of initial MVN morphologies. Finally, we found that MVNs exposed to flow have improved vascular barrier function and decreased platelet adhesion. MVNs with KLF2-based flow sensors represent a novel, powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.