In many tissues, cell shape and orientation are controlled by a combination of internal and external biophysical cues. Anisotropic substrate topography is a ubiquitous cue that leads to cellular elongation and alignment, a process termed contact guidance, whose underlying mechanisms remain incompletely understood. Additionally, whether contact guidance responses are similar in single cells and in cellular monolayers is unknown. Here, we address these questions in vascular endothelial cells (ECs) that in vivo form a monolayer that lines blood vessels. Culturing single ECs on microgrooved substrates that constitute an idealized mimic of anisotropic basement membrane topography elicits a strong, groove depth-dependent contact guidance response. Interestingly, this response is greatly attenuated in confluent monolayers. While contact guidance in single cells is principally driven by persistence bias of cell protrusions in the direction of the grooves and is surprisingly insensitive to actin stress fiber disruption, cell shape and alignment in dense EC monolayers are driven by the organization of the basement membrane secreted by the cells, which leads to a loss of interaction with the microgrooves. The findings of distinct contact guidance mechanisms in single ECs and in EC monolayers promise to inform strategies aimed at designing topographically patterned endovascular devices.

In many tissues, cell shape and orientation are controlled by a combination of internal and external biophysical cues. Anisotropic substrate topography is a ubiquitous cue that leads to cellular elongation and alignment, a process termed contact guidance whose underlying mechanisms remain incompletely understood. Additionally, whether contact guidance responses are similar in single cells and in cellular monolayers is unknown. Here, these questions in vascular endothelial cells (ECs) that in vivo form a monolayer that lines blood vessels are addressed. Culturing single ECs on microgrooved substrates that constitute an idealized mimic of anisotropic basement membrane (BM) topography elicits a strong, groove depth‐dependent contact guidance response. This response is principally driven by persistence bias of cell protrusions in the direction of the grooves and is surprisingly insensitive to actin stress fiber disruption. Interestingly, the contact guidance response is greatly attenuated in confluent monolayers, and cell shape and alignment in that case are driven by the organization of the BM secreted by the cells, which leads to a loss of cellular interaction with the microgrooves. The present finding of distinct contact guidance mechanisms in single ECs and in EC monolayers promises to inform strategies aimed at designing topographically patterned endovascular devices.