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

ABSTRACT: Background: Endothelial 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 clinically observed waveforms. Aim: To determine how the presence and temporal pattern of flow impact endothelial morphology and transcriptional activity in a 3D macrofluidic model. Methods: Idealised 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. The pulsatile waveform was derived from arteriovenous fistula Doppler ultrasound profiles and scaled to match mean volumetric flow (~100 mL/min) and time-averaged wall shear stress (~1.5 dyn/cm²) to the continuous-flow condition. Cell morphology was assessed using immunofluorescence. Bulk RNA-seq was performed (three independent biological replicates per condition) with Hallmark pathway enrichment using gene set enrichment analysis. Results: Relative to static culture, continuous flow increased cell eccentricity (0.74 vs 0.48, p <0.0001) and reduced variability in cell orientation (Δ = −47.1°, 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 reduced interferon signalling and the Hallmark inflammatory response program relative to static, while TNFα/NF-κB signalling was increased. Under matched mean shear, pulsatile versus continuous flow showed relative enrichment of cell-cycle/checkpoint programs in pulsatile flow (including mitotic spindle, G2M checkpoint, MYC targets V2, and E2F targets). Conversely, continuous flow showed relative enrichment of oxidative phosphorylation and the p53 pathway, whereas TGF-β signalling was relatively enriched in pulsatile flow. Conclusion: Under matched mean shear, pulsatile versus continuous flow were associated with distinct endothelial morphological and transcriptional signatures in HMEC-1. This vessel-scale 3D macrofluidic platform provides a validated, waveform-controlled testbed to support future mechanistic and translational studies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE328243 | GEO | 2026/08/19

REPOSITORIES: GEO

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Project description:d9 and d12 Mks were either cultured statically or subjected to shear flow for 30 min; at d9, half the Mks were placed back in culture for 30 min (60 min time point) Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation thus releasing platelets and pro/preplatelets directly into the blood stream. Shear forces have been now established as promoting Mk maturation and platelet biogenesis. In order to understand the underlying mechanisms that modulate the response of Mks to shear forces, we carried out transcriptional analysis on immature and mature stem cell-derived Mks that were exposed to physiologically-relevant shear (2.5 dyn/cm2). In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, 6 AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12. Our data show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD protein expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos protein expression. JNK inhibition reduced fibrinogen binding of d9 and d12 platelet-like particle s (PLPs) and P-selectin expression at d12 PLPs, while p38 inhibition reduced fibrinogen binding of d12 PLPs. Here we show that mechanotransduction of shear forces in Mks results in JNK activation, AP-1 upregulation, and downstream transcriptional changes that promote maturation of immature Mks and platelet biogenesis in mature Mks. Two- and Three-condition experiment (flow vs. static culture condition, d9 vs. d12, and 30 min vs. 60 min at d9); Biological replicates: 3; Technical replicates: 1 (dye-swap)

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

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