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

Abstract

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell phenotype 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 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 three-dimensionalin vitrosystems. Here we develop engineered microvascular networks (MVNs) with a KLF2-based endothelial cell 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 culture of MVNs exposed to flow for 48 hours that resulted in increased expression of the KLF2-GFP-reporter display larger vessel diameters and decreased vascular branching and resistance. Additionally, 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. The MVNs with KLF2-based flow sensors represent a powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2

“…Specifically, we used ECs carrying a flow-inducible reporter of KLF2 transcription [123] to construct threedimensional human MVNs within microfluidic devices and applied flow (or not) using an on-chip microfluidic pump, as in [124] (see also [125,126]).…”

“…We did not expose MVNs cultured under static conditions to flow. We provide further details about these protocols in [124]. See also [125,126].…”