Under pressure: Pressure loads can modify endothelial cell flow responses

Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the context of flow response is understudied, despite its known significance in vascular development and disease progression. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow. Our study demonstrates that elevated pressure conditions significantly modify shear-induced flow alignment and increase endothelial cell density, a phenomenon often observed in vascular diseases. Utilizing both bulk and single-cell RNA sequencing, we found that while flow is the primary driver of transcriptional changes from static conditions, pressure distinctly modulates this flow response by upregulating gene sets linked to arterial cell phenotypes. When compared to early vascular development stages, pressure was notably associated with gene sets that promote the formation of early hemogenic endothelial cells. Our findings emphasize the necessity of an integrative approach to endothelial cell mechanotransduction, one that encompasses the effects induced by pressure alongside other hemodynamic forces. Overall design: To investigate the effect of pressure conditions on endothelial cells responses to fluid shear stress, we cultured human pulmonary artery endothelial cells in microfluidic channels under different hemodynamic conditions. For static control conditions, cells were either cultured in a well plate (culture format = "wells") or in a microfluidic channel without flow. In all other samples, cells were exposed to controlled fluid shear stress (0, 5, 10, dyne/cm2) and pressure (0, 20, 60 mmHg) conditions for 24 hours to determine if pressure conditions modify the endothelial cell response to fluid shear stress.