Shear stress and very low levels of ligand synergize to activate ALK1 signaling in endothelial cells

Endothelial cells (ECs) respond to concurrent stimulation by biochemical fac-tors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemor-rhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. How-ever, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS mi-crofluidic device and a custom image analysis pipeline. We extended the previously reported syn-ergy between SS and BMP9, to include BMP10 and BMP9/10 . Moreover, we demonstrated this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm2) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1's basal activity and response to minimal ligand levels depend on endo-cytosis, distinct from cell-cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. Yet, an in-depth comprehension of ALK1 receptor trafficking's molecular mechanisms requires further investigation. ### Competing Interest Statement The authors have declared no competing interest.