AFM Force-to-Signal Assay Uncovers Glypican-1 and TRP Channel Roles in Endothelial Mechanotransduction

The endothelial surface glycocalyx (ESG) is a dense, carbohydrate-rich mesh of glycoproteins, proteoglycans, and glycosaminoglycans (GAGs) that coats the surface of endothelial cells (ECs). Mechanical cues, such as shear stress, at the ESG can be rapidly translated into biochemical signals. An impairment in this mechanosignaling pathway is linked to cardiovascular complications, such as diabetes, stroke, sepsis, and atherosclerosis, due to the decrease in the potent vasodilator, nitric oxide (NO). ESG plays a role in this pathway; however, the receptor-level pathways remain obscure. We present an atomic force microscopy (AFM) force-to-signal assay that delivers real-time, pico- to nano-newton, receptor-targeted mechanical inputs to a single live EC while simultaneously recording NO production via fluorescence. We apply controlled pulling forces to ESG components on mouse brain ECs to investigate which ESG elements are relevant to mechanosensing and which transient receptor potential (TRP) ion channels support Ca 2+ -dependent NO production. Our key findings are the following: (1) heparan sulfate (HS) and glypican-1 act as proximal mechano-sensors that trigger rapid NO signaling. (2) Pharmacological perturbation with SKF96365 markedly suppressed the mechanically evoked NO response, whereas a smaller but significant response persisted in the presence of amiloride. Together, our findings highlight glypican-1 engagement and the potential involvement of TRP-channel-associated Ca 2+ entry in the ESG-mediated mechanosignaling pathway. Additionally, by directly coupling a molecule-specific mechanical input to a quantitative fluorescent readout, this technique provides a direct, single-cell map of endothelial mechanotransduction from receptor to downstream signal, enabling exploration of relevant targets for cardiovascular pathologies associated with glycocalyx dysfunction.