Figure 3.
Leukocytes and flow synergistically induce PIEZO1 activation to stimulate endothelial downstream signaling. (A) HUVECs were preactivated with TNFα, loaded with Fluo-4, and exposed to PMNs alone, low flow (1.2 dynes/cm2) alone, or both. [Ca2+]i was determined as fluorescence intensity (RFU, relative fluorescence units). (B) HUVECs transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα, loaded with Fluo-4, and exposed to PMNs and low flow (1.2 dynes/cm2) given together. [Ca2+]i was determined as fluorescence intensity. Five traces representative of the traces of 1 experiment are shown in panels A and B, and the time point of addition of PMNs is indicated by an arrow. The bar diagrams in panels A and B show the area under the curve (AUC) of the [Ca2+]i traces from 6 independent experiments (20-40 cells were analyzed per experiment). (C-D) Currents from MLECs of wild-type (control) or EC-Piezo1-KO mice were recorded in the whole cell patch clamp configuration. The holding potential was −80 mV, and the MLECs were exposed to PMNs, low flow (1.2 dynes/cm2), or both (n = 8-9 independent measurements per condition). (E-H) Immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC in lysates of TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA directed against PIEZO1 and incubated without or with human PMNs in the absence or presence of low flow (1.2 dynes/cm2) (E) or without or with 5 μM Yoda1 (G). Immunoblot analysis of PIEZO1 and GAPDH served as controls. Bar diagrams (F,H) show the densitometric analysis of 3 independent experiments. (I) Transmigration of human PMNs across TNFα-activated HUVECs preincubated for 30 minutes with the PYK2 and SRC inhibitors PF431396 (10 μM) and PP2 (10 μM), respectively (n = 5 independent experiments). (J) HUVECs transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα and exposed to PMNs alone, low flow (1.2 dynes/cm2) alone, or both. After 15 minutes, VE-cadherin internalization was determined as described in Methods (n = 4 independent experiments). Shown are mean values ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001 (1-way ANOVA [A,D]; unpaired t test [B,F,H-J]).

Leukocytes and flow synergistically induce PIEZO1 activation to stimulate endothelial downstream signaling. (A) HUVECs were preactivated with TNFα, loaded with Fluo-4, and exposed to PMNs alone, low flow (1.2 dynes/cm2) alone, or both. [Ca2+]i was determined as fluorescence intensity (RFU, relative fluorescence units). (B) HUVECs transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα, loaded with Fluo-4, and exposed to PMNs and low flow (1.2 dynes/cm2) given together. [Ca2+]i was determined as fluorescence intensity. Five traces representative of the traces of 1 experiment are shown in panels A and B, and the time point of addition of PMNs is indicated by an arrow. The bar diagrams in panels A and B show the area under the curve (AUC) of the [Ca2+]i traces from 6 independent experiments (20-40 cells were analyzed per experiment). (C-D) Currents from MLECs of wild-type (control) or EC-Piezo1-KO mice were recorded in the whole cell patch clamp configuration. The holding potential was −80 mV, and the MLECs were exposed to PMNs, low flow (1.2 dynes/cm2), or both (n = 8-9 independent measurements per condition). (E-H) Immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC in lysates of TNFα-activated HUVECs transfected with control siRNA (siCtrl) or siRNA directed against PIEZO1 and incubated without or with human PMNs in the absence or presence of low flow (1.2 dynes/cm2) (E) or without or with 5 μM Yoda1 (G). Immunoblot analysis of PIEZO1 and GAPDH served as controls. Bar diagrams (F,H) show the densitometric analysis of 3 independent experiments. (I) Transmigration of human PMNs across TNFα-activated HUVECs preincubated for 30 minutes with the PYK2 and SRC inhibitors PF431396 (10 μM) and PP2 (10 μM), respectively (n = 5 independent experiments). (J) HUVECs transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) were preactivated with TNFα and exposed to PMNs alone, low flow (1.2 dynes/cm2) alone, or both. After 15 minutes, VE-cadherin internalization was determined as described in Methods (n = 4 independent experiments). Shown are mean values ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001 (1-way ANOVA [A,D]; unpaired t test [B,F,H-J]).

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