摘要：Atherosclerosis or fibrosis and cirrhosis undergo chronic inflammation associated with the adhesion between neutrophils and endothelial cells(ECs)that is mediated by their respective cellular adhesive molecules on stiffened blood vessel wall or extracellular matrix(ECM)under shear flow[1-3].However,the mechanical dependence of calcium flux and trail formation in neutrophils remains unclear yet in these processes.First,the effect of substrate stiffness through ECs on neutrophil calcium spike was quantified when the individual neutrophils adhered to EC monolayer pre-placed onto stiffness-varied polyacrylamide(PA)substrate(5 or 34.88 kPa)or glass surface.Our data indicated that E-/P-selectins and ICAM-1s on HUVECs and b2-integrins,PSGL-1s,and CD44s on neutrophils were all involved in mediating neutrophil calcium spike in a stiffness-dependent manner,in which the increase of substrate stiffness enhanced the calcium intensity and spike number.Such stiffness-dependent calcium response is associated with selectin-induced b2-integrin activation through Syk/Src signaling pathway and the F-actin/myosin II function.Moreover,tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike [4].Second,the trail formation of neutrophils to ECs monolayer pre-placed onto the same PA substrate were also tested under shear flow.Live fluorescence imaging showed that neutrophils are able to form long membrane tethers during migration and subsequently leave behind membranous long-lasting trails under shear,which are enriched in LFA-1,Mac-1,and CD44.Moreover,the formation of the trails was inhibited by blocking LFA-1s and Mac-1s,suggesting an important role forβ2-integrins in the trial formation.The recruitment of monocytes was inhibited when pre-blocking ICAM-1s on flowing monocytes,indicating that the neutrophil’s trails employβ2-integrin-ICAM-1 binding to recruit the monocytes.Intriguingly,both the length and the area of the trails increase with increasing substrate stiffness,result

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