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In a screen to identify endothelial transmembrane proteins involved in the transendothelial migration of leukocytes, we identified the mechanosensitive cation channel PIEZO1 (Figure 1A; supplemental Table 1, available on the Blood Web site). The siRNA-mediated knock down of PIEZO1 in HUVECs or in the mouse brain endothelial cell line bEnd.3 strongly reduced endothelial transmigration of PMNs and peripheral blood mononuclear cells (Figure 1B-D). Similarly, PMN transmigration through mouse lung endothelial cells (MLECs) from mice with endothelium-specific loss of Piezo1 (EC-Piezo1-KO)[28] was strongly reduced compared with wild-type MLECs (Figure 1E; supplemental Figure 1A-B). Basal and TNFα-induced expression of endothelial adhesion molecules was not affected by loss of PIEZO1 (supplemental Figure 1C). Both PMN transmigration of human andmurine endothelial cells could be stimulated by Yoda1, an activator of PIEZO1, and this effect was not seen after knockdown of PIEZO1 in endothelial cells (Figure 1D-G). PMN rolling on and adhesion to endothelial cells was not affected by loss of endothelial Piezo1 expression (Figure 1B-C), and endothelial barrier function analyzed by measuring the electrical impedance of the endothelial cell layer in vitro or by determining the permeability of the endothelial layer for FITC-labeled dextran in vivo was normal after loss of PIEZO1 (Figure 1H-I).
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EnlargeFigure 1. PIEZO1 mediates leukocyte transendothelial migration in vitro. (A) HUVECs pretreated with 10 ng/mL TNFα were transfected with 360 siRNAs pools against RNAs encoding transmembrane proteins expressed in endothelial cells and were then exposed to THP-1 monocytic cells for 3 hours. Shown is the ratio of THP-1 cells that transmigrated the HUVEC monolayer transfected with a particular siRNA pool and with control siRNA. The plot shows the ranked average ratios of 3 independent experiments. (B) HUVECs were transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1), and rolling, adhesion, and transmigration of human PMNs applied together with flow (1.2 dynes/cm2) were analyzed (n = 8 independent experiments per group). Cells treated with control siRNA were set as 100%. (C-I) The indicated endothelial cells were transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) or were left untransfected (E). (C) Rolling, adhesion, and transmigration of mouse PMNs (n = 8 per group) applied together with flow (1.2 dynes/cm2) to a bEnd.3 cell monolayer. Cells treated with control siRNA were set as 100%. (D,F,G) Transmigration of human peripheral blood mononuclear cells (D) (n = 4 independent experiments per group), human PMNs (F) (n = 6 independent experiments per group), or mouse PMNs (G) (n = 6 independent experiments per group) across HUVECs (D,F) or bEnd.3 cells (G) pretreated without or with 1 μM Yoda1 for 15 minutes. (E) MLECs were isolated from EC-Piezo1-KO and control mice, and transmigration of mouse PMNs was determined after pretreament without or with 1 μM Yoda1 for 15 minutes (n = 5 independent experiments). (H) HUVEC barrier integrity was assessed using an electric cell-substrate impedance sensing (ECIS) system in the absence or presence of 1 μM Yoda1 (n = 8 independent experiments per group). (I) Paracellular permeability of the endothelial monolayer cultured in transwell plates was determined using 40 kDa FITC-dextran (n = 5 independent experiments per group; a.u., arbitrary units). Shown are mean values ± SEM; *P ≤ .05; **P ≤ .01; ***P ≤ .001 (unpaired t test [B-H], 2-way ANOVA [I]).
To study leukocyte extravasation in vivo, we injected TNFα into the peritoneal cavity and determined the number of CD11b+/Ly6G+ myeloid cells in the peritoneal cavity 6 hours later. Whereas TNFα induced a significant influx of cells into the peritoneal cavity of wild-type mice compared with untreated controls, the effect of TNFα was strongly reduced in EC-Piezo1-KO mice (Figure 2A). We then studied the role of endothelial PIEZO1 in a model of acute dermatitis of the ear by applying croton oil to the ear surface and found that the total number of neutrophils seen in sections of ears from wild-type and EC-Piezo1-KO mice was identical (supplemental Figure 1D). However, when analyzing postcapillary venules characterized by a diameter of 20 to 30 µm, the primary site of leukocyte extravasation, we found that most neutrophils had completed extravasation and were found in the perivascular space in wild-type mice, whereas about 25% to 30% of the leukocytes were found in the lumen of vessels (Figure 2B-D). However, in EC-Piezo1-KO mice, a significantly reduced portion of leukocytes had completed extravasation, and the majority, about 70% of cells, showed arrest at the luminal surface of the endothelium (Figure 2B,D), suggesting that they adhered to the endothelium but were not able to initiate the process of endothelial transmigration. Similarly, LPS-induced extravasation of neutrophils into the lung parenchyma was strongly reduced in EC-Piezo1-KO mice (Figure 2E-F). Staining and intravital microscopy of the cremaster of EC-Piezo1-KO mice revealed a reduced extravasation of neutrophils compared with controls after intrascrotal injection of interleukin 1β (IL-1β; Figure 2G-I). Hemodynamic parameters were similar in both mouse types, and there was no significant different in leukocyte rolling and adhesion within venules (supplemental Figure E-I). Also, basal extravasation of Evans blue and extravasation after subcutaneous injection of histamine or vascular endothelial growth factor (VEGF) were indistinguishable between wild-type and EC-Piezo1-KOmice (Figure 2J), indicating that vascular permeability was unchanged. Expression of genes encoding proteins involved in endothelial functions was not changed in endothelial cells from EC-Piezo1-KO mice (supplemental Figure 1J-K).
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EnlargeFigure 2. PIEZO1 mediates leukocyte transendothelial migration in vivo. (A) Endothelium-specific PIEZO1-deficient mice (EC-Piezo1-KO) or control animals were injected intraperitoneally with PBS or 500 ng of TNFα, and the number of peritoneal CD11b+;Ly6G+ neutrophils was determined by flow cytometry (n = 4 mice, −TNFα; n = 5 mice, +TNFα). (B-D) EC-Piezo1-KO and control mice were treated with croton oil on 1 ear. Six hours later, animals were euthanized, and ears were immunostained as whole mounts with antibodies against PECAM-1 (blue, endothelium), collagen-IV (red, basement membrane), and MRP14 (green, neutrophil). Arrows indicate neutrophils. Scale bar, 10 μm. (B) Representative images of stained ears. (C) Schematic drawing illustrating the criteria to delineate the 5 positions in which leukocyte are found during extravasation. (D) Distribution pattern of neutrophil positions relative to the endothelium and basement membrane (n = 16 mice, control; n = 14 mice, EC-Piezo1-KO; 3–5 vessels were analyzed per animal). (E-F) Confocal imaging of PMNs and lung microvessels 4 hours after intraperitoneal injection of 1 mg/kg LPS in EC-Piezo-KO and control mice (E). Quantitative analysis of extravasated neutrophils per field (F; n = 6 mice, wild type; n = 6 mice, EC-Piezo1-KO). (G-H) EC-Piezo1-KO and control animals were injected with 50 ng (in 100 μL PBS) IL-1β intrascrotally. After 3 hours, the cremaster muscle was isolated and stained for PECAM-1 and MRP14 (G). The quantitative analysis of extravasated neutrophils per vessel area is shown in panel H (n = 8 mice, control; n = 10 mice, EC-Piezo1-KO; 2–3 vessels were analyzed per animal). (I) EC-Piezo1-KO and control mice were analyzed by intravital microscopy of cremaster venules 4 hours after injection of 50 ng IL-1β for extravasated leukocytes (n = 9 mice per group; 4–10 measurements per animal). (J) Evans blue extravasation was assessed after subcutaneous injection of 20 μL PBS without or with 100 μM of histamine or 100 ng/mL VEGF (n = 8 mice, PBS and histamine; n = 4 mice, VEGF). Shown are mean values ± SEM. n.s., nonsignificant; **P ≤ .01; ***P ≤ .001 (unpaired t test).
Because increases in [Ca2+]i are involved in the initiation of leukocyte transendothelial migration and because leukocyte diapedesis occurs in the presence of low flow in vivo, we studied leukocyte-induced increases in endothelial cytosolic Ca2+ in the absence and presence of flow at a low shear rate (1.2 dynes/cm2). In control HUVECs loaded with the [Ca2+]i indicators Fluo-4 or Fura-2, low flow alone or addition of human neutrophils alone had only a small effect on the cytosolic [Ca2+] (Figure 3A; supplemental Figure 2A). Similar but less pronounced effects were seen with the even lower shear rates of 0.4 and 0.8 dynes/cm2 (supplemental Figure 2B). However, when neutrophils were given together with flow, we observed a strong increase in the endothelial cytosolic Ca2+ concentration (Figure 3A; supplemental Figure 2A). This synergistic effect was rarely seen during rolling or initial arrest of leukocytes but during crawling and during the transmigration phase (supplemental Figure 2C), and it depended on PIEZO1 (Figure 3B; supplemental Figure 2D). Chelation of intra- and extracellular Ca2+ by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and EGTA, respectively, blocked Ca2+ transients (supplemental Figure 2E-F). Whole cell patch-clamp recordings of MLECs showed characteristic inward currents when cells were exposed simultaneously to low flow and PMNs, which were not seen in MLECs from EC-Piezo1-KO animals (Figure 3C-D; supplemental Figure 2G-H). In contrast, exposure of MLECs to flow alone or PMNs alone induced only small currents (Figure 3C-D).
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EnlargeFigure 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]).
We then tested the potential involvement of PIEZO1 in the induction of downstream signaling events mediating leukocyte-induced opening of endothelial junctions. Whereas low flow alone had hardly any effect on the phosphorylation of PYK2, SRC, and the myosin light chain (MLC) in endothelial cells, addition of PMNs had as small but significant effect. However, application of both flow and PMNs synergistically induced endothelial PYK2, SRC, and MLC phosphorylation to a degree significantly higher than each of the 2 stimuli alone, and this effect was strongly reduced after knockdown of PIEZO1 (Figure 3E-F). The effect of PMNs and flow was mimicked by application of Yoda1, and this effect was again blocked after knockdown of PIEZO1 (Figure 3G-H). Inhibition of endothelial PYK2 or SRC by PF431396 or PP2, respectively, reduced basal transmigration and blocked Yoda1-induced increases in PMN transmigration (Figure 3I). These data strongly indicate that PMNs and low flow synergistically induce downstream signaling events through endothelial PIEZO1, resulting in the opening of endothelial junctions and leukocyte transmigration. Consistent with this, we also observed synergism in the ability of flow and PMNs to induce internalization of vascular endothelial (VE)-cadherin, an effect strongly inhibited after siRNA-mediated suppression of Piezo1 expression (Figure 3J).
Because engagement of endothelial ICAM-1 by leukocyte β2 integrins is essential for induction of increases in [Ca2+]i and diapedesis,[13,15,29,30] we suppressed expression of endothelial ICAM-1 and found that this strongly inhibited PMN-induced Ca2+ transients and PYK2, SRC, and MLC phosphorylation (Figure 4A-B; supplemental Figure 3A-B). Clustering of ICAM-1 using beads coated with anti–ICAM-1 antibodies mimicked the effect of PMNs and induced Ca2+ transients and phosphorylation of PYK2, SRC, and MLC synergistically with low flow (Figure 4C-D; supplemental Figure 3C), whereas beads coated with a control IgG had no effect (supplemental Figure 3D-E). The effects of ICAM-1 clustering were inhibited after knockdown of PIEZO1 and ICAM-1 (Figure 4C-D). Similar results were obtained when ICAM- 1 clustering was induced by cross-linking of bound anti–ICAM-1 antibodies (supplemental Figure 3F-I). When beads coated with anti–ICAM-1 antibodies were given together with low flow, an inward current was induced, which was sensitive to Gd3+ and the PIEZO1 inhibitor GsMTx4, whereas beads and flow alone had hardly any effect (Figure 4E-F). This strongly indicates that clustering and activation of ICAM-1 by leukocytes in the presence of low flow results in PIEZO1-mediated downstream signaling leading to the opening of endothelial junctions.
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EnlargeFigure 4. 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]).
ICAM-1 clustering and adhesion of leukocytes to endothelial cells have been shown to induce stiffening of the endothelial surface and to induce traction stress.[31–35] We therefore determined membrane tension in response to ICAM-1 clustering and PMNs using the fluorescent lipid tension sensor FliptR[36] and the membrane stress sensor (MSS) biosensor[35] (supplemental Figure 4A-B). We found that clustering of ICAM-1 or addition of PMNs leads to a small increase in endothelial membrane tension (Figure 5A-D). Low flow, which by itself had no significant effect on endothelial membrane tension, when given together with ICAM-1 clustering agents or PMNs, resulted in a very strong increase in plasma membrane tension (Figure 5A-D). This indicates that low flow and ICAM-1 clustering synergistically increase endothelial membrane tension.
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EnlargeFigure 5. Flow and ICAM-1 clustering synergistically increase endothelial membrane tension. (A-B) Fluorescence lifetime τ1 images of FliptR in TNFα-activated HUVECs kept under static conditions or in the presence of low flow (1.2 dynes/cm2) or exposed to anti–ICAM-1 antibody beads or to anti–ICAM-1 crosslinking antibodies (ICAM-1 XL) without or together with low flow. The color bar corresponds to lifetime in nanoseconds. Scale bar, 15 μm. Corresponding lifetime mean values indicating membrane tension are shown in the bar diagram (B; n = 40 measurements from 5 independent experiments). (C-D) Representative pseudocolored Förster resonance energy transfer (FRET) images of MSS-expressing cells kept under static conditions or in the presence of low flow (1.2 dynes/cm2) or exposed to anti–ICAM-1 crosslinking antibodies (ICAM-1 XL) without or together with low flow (C). The color bar indicates YPet/ECFP emission ratio. Corresponding normalized YPet/ECFP emission ratio of Förster resonance energy transfer (FRET) biosensors indicating membrane tension are shown in the bar diagram (D; n = 15–22 measurements from 3 independent experiments). Shown are mean values ± SEM. n.s., nonsignificant; **P ≤ .01; ***P ≤ .001 (1-way ANOVA).
Because ICAM-1 clustering has been shown to induce localized actin polymerization, MLC phosphorylation, and actomyosin contractility, which promote junctional opening,[18,31] we analyzed the effect of cytochalasin D and blebbistatin on membrane tension and on phosphorylation of PYK2, SRC, and MLC induced by ICAM-1 clustering. Both agents blocked ICAM-1–dependent changes in membrane tension and downstream signaling (Figure 6A-D). We then tested whether increased membrane tension and downstream signaling induced by ICAM-1 clustering involves the actin adapter proteins α-actinin-4 and cortactin, which have been shown to be recruited after clustering of ICAM-1 and be required for ICAM-1–mediated actin filament branching and for ICAM-1–dependent transendothelial migration of neutrophils.[37–39] As shown in Figure 6E-G and supplemental Figure 5A-B, siRNA-mediated knockdown of the RNAs encoding β-actinin-4 and cortactin blocked the effect of ICAM-1 clustering on membrane tension and downstream signaling. These data suggest that actin polymerization and actomyosin contractility of the cortical cytoskeleton induced by ICAM-1 clustering and leading to increased cortical tension[40,41] directly affect plasma membrane tension[41] and thereby induce PIEZO1 activation.
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EnlargeFigure 6. Actin polymerization and actomyosin contractility mediate increase of endothelial membrane tension and downstream signaling induced by ICAM-1 clustering. (A-G) HUVECs were preincubated without or with 10 μM cytochalasin D (CytoD) or 30 mM blebbistatin (Bleb) (A-D) or were transfected with control siRNA (siCtrl) or siRNA directed against the RNA encoding α-actinin-4 (siACTN4) or cortactin (siCTTN) (E-G) and were exposed to low flow alone, anti–ICAM-1 clustering antibodies (ICAM-1 XL) alone or both, and membrane tension was determined using FliptR (A-B,G; n = 20 measurements from 3 independent experiments), or immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC was performed (C-F). Bar diagrams show lifetime mean values (B,G) or immunoblot analysis of total and phosphorylated PYK2, SRC, and MLC (D,F; 3 independently performed immunoblot experiments). (H) Schematic representation showing how fluid shear stress exerted by the flowing blood and leukocyte-induced ICAM-1 clustering synergistically activate PIEZO1 to induce downstream signaling events resulting in opening of the endothelial barrier. Shown are mean values ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001 (1-way ANOVA).
