Shear Difference: Flow Type Dictates Endothelial Flow‐Responsive Gene Programs in a 3‐Dimensional–Printed In Vitro Model

Background Endothelial cells are mechanosensitive and adopt distinct phenotypes in response to hemodynamic forces. These responses are difficult to study in conventional in vitro platforms, which rarely reproduce vessel‐scale geometry or clinically relevant flow. Our aim was to determine how flow impacts endothelial morphology and transcriptional activity in a 3‐dimensional macrofluidic model. Methods Idealized vessels were 3‐dimensional printed using a water‐soluble polyvinyl alcohol and cast in polydimethylsiloxane. After core dissolution, human microvascular endothelial cell line‐1 cells were grown on the lumen and perfused for 24 hours under static, continuous, or pulsatile flow. The pulsatile waveform was derived from arteriovenous fistula Doppler profiles and scaled to match mean volumetric flow (~100 mL/min) and time‐averaged wall shear stress (~1.5 dyn/cm 2 ) to continuous flow. Morphology was assessed using immunofluorescence. Bulk RNA‐sequencing and gene set enrichment analysis were performed. Results Relative to static culture, continuous flow increased cell eccentricity (0.74 versus 0.48; P <0.0001) and reduced orientation variability (Δ=−47.1°, P <0.0001). Differential gene expression was extensive (continuous versus static: 2103 genes; pulsatile versus static: 2643 genes; pulsatile versus continuous: 384 genes). Continuous flow reduced interferon signaling and the Hallmark inflammatory response program relative to static, whereas tumor necrosis factor‐α/nuclear factor‐κ–light‐chain enhancer of activated B cells signaling was increased. Under matched mean shear, pulsatile flow enriched cell cycle/checkpoint programs. Conversely, continuous flow enriched oxidative phosphorylation and p53 pathways. Transforming growth factor‐β signaling was enriched in pulsatile flow. Conclusions Under matched mean shear, pulsatile and continuous flow were associated with distinct endothelial morphologic and transcriptional signatures. This macrofluidic platform provides a validated, waveform‐controlled testbed for mechanistic and translational studies.