Endothelial PIEZO1 activation by leukocytes involves ICAM-1 activation and flow. (A-B) HUVECs were transfected with control siRNA (siCtrl) or siRNA directed against ICAM-1. After treatment with TNFα, cells were exposed to low flow and human PMNs (A) or to flow and PMNs alone or given together (B). Thereafter, the free [Ca²⁺]_i was determined after loading of HUVECs with Fluo4 (A), or immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC was performed (B). Immunoblot analysis of GAPDH served as control. The bar diagram (A) shows the AUC of the [Ca²⁺]_i-trace from 3 independent experiments. The bar diagram (B) shows the densitometric analysis of 3 independent experiments. The arrow in panel A indicates the time point of addition of PMNs. (C-D) TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA directed against ICAM-1 or PIEZO1 were exposed to low flow and anti–ICAM-1 antibody beads (ICAM-1 beads) given together (C) or to low flow and anti–ICAM-1 beads alone or given together (D). Thereafter, the free [Ca²⁺]_i was determined after loading of HUVECs with Fluo-4 (C), or immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC (D) was performed. Traces shown in panel C represent signals from 20 to 40 cells, and the time point of addition of beads is indicated by an arrow. In panel D, immunoblot analysis of GAPDH served as controls. Bar diagrams (C) show the AUC of the [Ca²⁺]_i traces from 3 independent experiments. Bar diagrams (D) show the densitometric analysis of 3 independent experiments. (E-F) Currents from HUVECs pretreated without or with 10 µM Gd³⁺ or 5 µM GsMTx4 and exposed to low flow (1.2 dynes/cm²), anti–ICAM-1 beads, or both were recorded in the whole cell patch clamp configuration at a holding potential of −80 mV. Shown are characteristic traces (E) and statistical analysis of 5 to 12 independent recordings (F). Shown are mean values ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001 (unpaired t test [A-B]; 1-way ANOVA [C-D,F]).