Mechanical features of endothelium regulate cell adhesive molecule-induced calcium response in neutrophils.

Atherosclerosis is caused by chronic inflammation associated with the adhesion of neutrophils and endothelial cells (ECs) that is mediated by their respective cellular adhesive molecules to stiffened blood vessel walls. However, the stiffness dependence of calcium flux on neutrophils remains unclear yet. Here, the effect of substrate stiffness by ECs on neutrophils' calcium spike was quantified when the individual neutrophils that adhered to the human umbilical vascular endothelial cell (HUVEC) monolayer were pre-placed onto a stiffness-varied polyacrylamide substrate (5 or 34.88 kPa) or glass surface. Our data indicated that E-/P-selectins and intercellular adhesion molecule 1 (ICAM-1) on HUVECs and β2-integrins, P-selectin glycoprotein ligand 1 (PSGL-1), and CD44s on neutrophils were all involved in mediating neutrophil calcium spike in a stiffness-dependent manner, in which the increase in substrate stiffness enhanced the calcium intensity and the oscillation frequency (spike number). Such stiffness-dependent calcium response is associated with the induced selectin related to β2-integrin activation through the Syk/Src signaling pathway, and F-actin/myosin II are also involved in this. Moreover, tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike. These results provide an insight into understanding how the stiffening of vascular walls could regulate the calcium flux of adhered neutrophils, and thus the immune responses in atherosclerosis.