Shear Difference: Flow Type Dictates Endothelial Flow-Responsive Gene Programs in a 3D-Printed in vitro Model

BackgroundEndothelial cells (ECs) are mechanosensitive and adopt distinct phenotypes in response to hemodynamic forces. These phenotypes are rarely seen in shear-responsive signalling studies because commonly used in vitro platforms rarely reproduce the true scale of vessel geometries or physiologic waveforms. AimTo determine how the presence and temporal pattern of flow impact endothelial morphology and transcriptional activity in a 3D macrofluidic model. MethodsIdealised vessels were 3D-printed using a water-soluble polyvinyl alcohol filament and cast in polydimethylsiloxane. The polyvinyl alcohol cores were dissolved leaving a polydimethylsiloxane lumen on which HMEC-1 cells were grown and perfused for 24 h under static, continuous flow, or pulsatile flow. Cell morphology was assessed using immunofluorescence. Bulk RNA-seq was performed with Hallmark pathway enrichment using gene set enrichment analysis. ResultsRelative to static culture, continuous flow increased cell eccentricity (0.74 vs 0.48, p <0.0001) and reduced variability in cell orientation ({Delta} = -47.1{degrees}, p <0.0001). At the transcriptome level, differential gene expression was extensive (continuous vs static: 2,103 genes; pulsatile vs static: 2,643 genes; pulsatile vs continuous: 384 genes). Continuous flow favoured oxidative-metabolic and barrier-maintenance pathways. By contrast, pulsatile shear of the same mean load promoted cell-cycle/checkpoint signalling rather than oxidative-metabolic programmes. Head-to-head, pulsatile flow emphasized MYC proto-oncogene (MYC) and E2F transcription factor (E2F), whereas continuous flow preferentially engaged oxidative phosphorylation and phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase (AKT)-mechanistic target of rapamycin (MTOR) (with higher tumor protein p53and transforming growth factor-{beta} signaling). ConclusionUnder matched mean shear, pulsatile versus continuous flow drive distinct endothelial morphological and transcriptional programs, enriching pathways central to blood vessel function, remodelling, and disease. 3D macrofluidic platforms can replicate flow-specific endothelial cell mechanobiology, providing a translational tool to better understand vascular outcomes.