Discussion

We report that the mechanosensitive cation channel PIEZO1 plays a critical role in transendothelial migration of leukocytes in vitro and in vivo by integrating low levels of fluid shear stress and mechanical signals induced by clustering of ICAM-1 and thereby mediating increases in [Ca²⁺]_i and a localized opening of the endothelial barrier (Figure 6H). It has been reported that TRPC6 is critically involved in leukocyte-induced increases in endothelial [Ca²⁺]_i during leukocyte transendothelial migration.^[19] In expression analyses, we were not able to observe expression of TRPC6 in endothelial cells (supplemental Figure 1J). Nevertheless, it could well be that both PIEZO1 and TRPC6 operate in parallel under in vivo conditions or that PIEZO1 is involved in the initiation of leukocyte extravasation by sensing both flow and leukocyte adhesion, whereas TRPC6 mediates increases in [Ca²⁺]_i mainly at later stages of diapedesis.

ICAM-1 is a central endothelial adhesion receptor that functions as a ligand for β2 integrins on leukocytes and promotes leukocyte spreading, migration, and transmigration.^[29,42] Engagement of ICAM-1 leads to the clustering of ICAM-1 molecules and its redistribution into ring-like structures around adherent leukocytes, which is a requirement for efficient downstream signaling.^[43,44] ICAM-1 clustering induces cytoskeletal changes such as actin polymerization, MLC phosphorylation, and actomyosin contractility, which promote junctional opening.^{[18,31,42,45]} ICAM-1 also promotes increase in [Ca²⁺]_i levels,^[13,46] which has been shown to lead to activation of SRC via protein kinase C.^[15] ICAM-1–mediated activation of SRC and PYK2 has been shown to be required for VE-cadherin–dependent leukocyte transendothelial migration.^[30] This involves direct phosphorylation of VE-cadherin^[30,47,48] and indirect regulation of VE-cadherin through VE-PTP^[49] or by phosphorylation of β-catenin.^[50] How ICAM-1 clustering induces activation of these downstream signaling events resulting in junctional opening and transendothelial migration is poorly understood. Our data indicate that downstream signaling through ICAM-1 requires coactivation of PIEZO1 by fluid shear stress and ICAM-1–induced reorganization of the cortical cytoskeleton.

Various mechanical stimuli acting on cellular membranes have been shown to be able to activate PIEZO1. These include exposure to fluid shear stress, mechanical indentation of the cell surface, cell migration, compression of the cell membrane, or forces generated at the cell–cell or cell–matrix interface.^[21,51] Our data show that low-level fluid shear stress and interaction of leukocytes with the endothelial surface act in a synergistic manner to activate endothelial PIEZO1 and to initiate leukocyte transendothelial migration. In postcapillary venules, the place where leukocyte extravasation mainly takes place, the shear stress exerted by the flowing blood is relatively low at about 1 to 2 dynes/cm,^[2,52,53] a shear rate hardly able to induce PIEZO1-mediated signaling.^[27,28] Consistent with this, we saw only very small increases in [Ca²⁺]_i and no significant increase in the phosphorylation of PYK2, SRC, or MLC in response to fluid shear stress of 1.2 dynes/cm². Similarly, when ICAM-1 clustering was induced in TNFα-pretreated endothelial cells by PMNs or anti–ICAM-1 antibodies, only small increases in [Ca²⁺]_i and phosphorylation of PYK2, SRC, and MLC could be observed, which were further reduced after suppression of Piezo1 expression. However, when endothelial ICAM-1 clustering was induced while exposing cells to low flow, downstream signaling was strongly activated, and this effect was inhibited by knockdown of PIEZO1. This raised the question as to how ICAM-1 clustering promotes PIEZO1 activation. Both 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.^[31–34,54] These endothelial responses are caused by increased actin polymerization and actomyosin contractility of the cortical cytoskeleton, which lead to increased cortical tension^[40,41] and require recruitment of the actin adapter proteins a-actinin-4 and cortactin.^[31,37] Because the plasma membrane and the underlying cortical cytoskeleton are closely interconnected,^[40,41] changes in the actomyosin cortical tension directly affect plasma membrane tension^[41] and therefore are likely to regulate PIEZO1 activity. Consistent with this, we found that inhibition of actin polymerization and myosin activity and siRNA-mediated knockdown of α-actinin-4 and cortactin blocked ICAM-1–mediated increases in membrane tension and PIEZO1-dependent downstream signaling required for leukocyte transendothelial migration. We therefore also think that it is the combined effect of low flow and leukocyte-induced ICAM-1 clustering on plasma membrane tension that induces PIEZO1 activation. This is consistent with a series of biophysical studies showing that changes in plasma membrane tension are sufficient to regulate the open probability of PIEZO1 and that no intra- or extracellular interactions are required.^[23–25] However, we cannot exclude that additional mechanisms are involved. For instance, it has recently been suggested that Piezo1 can be linked to the actin cytoskeleton through members of the cadherin family including VE-cadherin,^[55] and this mechanism may contribute to Piezo1 activation induced by ICAM-1 clustering and subsequent actin polymerization.

Recent data indicate that changes in plasma membrane tension are restricted to subcellular domains of endothelial cells as local increases in membrane tension lead only to local activation of mechanosensitive ion channels such as PIEZO1.^[56] The finding that leukocyte-induced endothelial downstream signaling and diapedesis require PIEZO1 and flow is consistent with earlier observations, which showed that fluid shear stress promotes transendothelial leukocyte migration.^[57–60] Our data identify a novel synergism of local hemodynamic forces and initial endothelial leukocyte adhesion to induce plasma membrane tension and endothelial signaling events that promote leukocyte extravasation. The discovery of a novel mechanosensing and mechanosignaling process required for the initial phase of leukocyte diapedesis may also lead to new anti-inflammatory therapeutic approaches.