Methods

Immunoblot Analysis

Cells were lysed in cell lysis buffer (9803; Cell Signaling) containing 1% triton X-100 or in radioimmunoprecipitation assay (RIPA) buffer (9806; Cell Signaling) supplemented with protease and phosphatase inhibitors (5872; Cell Signaling). Lysates were centrifuged at 10 000g at 4°C for 10 minutes. Supernatants were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with primary and horseradish peroxidase–conjugated secondary antibodies (8884 and 7076, respectively; Cell Signaling) and were developed using the enhanced chemiluminescence (ECL) detection system (Pierce).

Determination of [Ca²⁺]_i

For the determination of the intracellular Ca²⁺ concentration, endothelial cells were loaded with 5 μM Ca²⁺-sensitive dye Fluo-4 AM (F14201; Molecular Probes) or 5 μM Fura-2 AM (F1221; Molecular Probes) in Hanks balanced salt solution supplemented with 20 mM N-2-hydroxyethylpiperazine-N'–2-ethanesulfonic acid (HEPES) for 30 minutes at 37 °C and were then washed with Hanks balanced salt solution 3 times at room temperature. Live-cell images were acquired with an IX81 microscope (Olympus) at a frequency of 1 Hz. Fluo-4 fluorescence was measured by using excitation at 488 nm and emission collected at 500 to 550 nm. Fura-2 was monitored by digital fluorescence 340/380-nm ratio imaging. In experiments in which polymorphonuclear leukocytes (PMNs) were added to endothelial cells, only endothelial cells to which PMNs were attached were used for the analysis.

Fluorescein Isothiocyanate-dextran Permeability Assay

A total of 1.5 × 10⁴ human umbilical venous endothelial cells (HUVECs) were seeded per well of a collagen-coated transwell plate (3-μm pore size; Corning) and were cultured with daily medium changes until reaching confluency. For knockdown experiments, 8000 cells were transfected using Lipofectamine RNAiMAX with the indicated small interfering RNAs (siRNAs). For permeability assay, the medium of the upper insert was removed and replaced with medium containing 250 μg/mL fluorescein isothiocyanate (FITC)-conjugated dextran (relative molecular mass, 40 kDa; Molecular Probes). The permeability was determined by passage of FITC-dextran through the endothelial monolayer into the lower chamber using FlexStation-3 (Molecular Devices).

Electrophysiology

Whole cell patch-clamp recordings were performed at room temperature using an EPC10/2 amplifier with Pulse software (HEKA Electronik GmbH, Lambrecht, Germany). Pipette resistance was between 3 and 4 MΩ, and membrane potential was clamped at −80 mV. Normal external solution contained 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1mM MgCl_2, 10 mM HEPES, and 10 mM glucose, pH 7.4. The intracellular pipette solution contained 95 mM L-aspartate, 40 mM CsCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 0.1 mM guanosine triphosphate, pH 7.2. Local low flow at indicated shear rate was generated with a multichannel microperfusion system (LEAD-2, LONGER). Mechanical stimulation was applied to cells using a fire-polished glass pipette with a 3- to 5-μm tip diameter. Pipette movement was controlled by PCS-5000 Patch-Clamp micromanipulator (Burlegh). The micromanipulator uses the solid-state stability of piezoelectric technology to maintain smooth and predictable pipette motion without drift. Whole cell current was analyzed with IGOR Pro software (WaveMetrics). Experiments were performed with dissociated single cells. In experiments in which PMNs were added to endothelial cells, only endothelial cells to which PMNs were attached were used for the analysis.

PMN Application and ICAM-1 Clustering

We routinely applied 2 × 10⁵ PMNs per milliliter to cells. For antibody-mediated clustering of ICAM-1, sheep anti-mouse IgG-coupled dynabeads (M280; Invitrogen) were coated with mouse anti-human ICAM-1 antibody (BBIG-I1; R&D Systems) or immunoglobulin IgG1 (IgG1) control (MAB002; R&D Systems) overnight at 4°C according to the manufacturer's protocol. To induce clustering, 1.5 × 10⁶ antibody-coated beads/mL was added to a tumor necrosis factor a (TNFα)-stimulated HUVEC monolayer cultured in a 6-well dish and incubated for 15 minutes. For some experiments, antibody-coated beads (5 × 10⁵/mL) were injected into the ibidi perfusion system containing HUVECs to induce ICAM-1 clustering on HUVECs under physiologic flow conditions. Alternatively, ICAM-1 was ligated with 15 μg/mL mouse monoclonal antibodies (R&D Systems, catalog no. BBIG-I1) for 30 minutes, followed by washing and ICAM-1 cross-linking with 50 μg/mL mouse secondary antibody (R&D Systems, catalog no. AF007) for 20 minutes at 37°C. Live cell imaging of membrane tension and intracellular Ca²⁺ during ICAM-1 clustering were performed using a Leica SP8 or an Olympus IX81 microscope (see above). For immunoblot analysis, beads were isolated using a magnetic holder (Miltenyi Biotec), and cells were lysed with RIPA buffer as described above.

Lipopolysaccharide (LPS)-induced Extravasation

EC-Piezo1-knockout (KO) and control mice were injected intraperitoneally with 200 μL phosphate-buffered saline (PBS) alone or containing 1 mg/kg body weight lipopolysaccharide (LPS) (L4391; Sigma). After 4 hours, animals were euthanized, and lungs were collected for immunostaining with anti-MPR14 and anti–platelet endothelial cell adhesion molecule 1 (PECAM-1) antibodies. For quantification of extravasating PMNs, cryosections were analyzed in XYZ views on a Leica SP8 confocal microscope by the following criteria: PMNs directly surrounded by PECAM-1 staining (ie, blood vessel) were scored as intravascular, whereas cells outside blood vessels were scored as extravascular.

TNFα-induced Peritonitis and Flow Cytometry

Wild-type or EC-Piezo1-KO mice were injected intraperitoneally with 100 μL PBS containing no or 500 ng TNFα prewarmed to 37°C. After 60 minutes, animals were euthanized, and cells in the peritoneal cavity were collected by flushing with 5 mL ice-cold PBS. Peritoneal cells were filtered using a 70-μm strainer and analyzed by flow cytometry (BD FACS Canto II). The following antibodies were used: FITC conjugated anti-mouse CD11b (101205; BioLegend) and allophycocyanin (APC)-conjugated anti-mouse Ly6G (127614; Biolegend).

Other Reagents and Antibodies

Yoda1 (5586) was from Tocris Bioscience. Cytochalasin D (C8273), Blebbistatin (B0560), and PF431396 (PZ0185) were from Sigma. PP2 (529576) was from Merck Chemicals. Anti–PIEZO1-antibody was from Proteintech (15939-1-AP). Anti-GAPDH (catalog no. 5174), anti-protein tyrosine kinase 2 (PYK2) (catalog no. 3292), anti–p-PYK2 (Tyr402; 3291), antisarcoma (SRC) (2109), anti–p-SRC (Tyr416; 6943), anti-MLC (3672), and anti–p-MLC (Ser19; 3675) were from Cell Signaling Technology. Anti–endomucin antibody was from Santa Cruz (sc-65495). Gd³⁺ (439770) was from Sigma, and GsMTx4 (4912) was from Tocris.

Statistical Analysis

Trial experiments or experiments done previously were used to determine sample size with adequate statistical power. Samples were excluded in cases where RNA/cDNA quality or tissue quality after processing was poor (below commonly accepted standards). Data are presented as means ± standard error of the mean (SEM). Comparisons between 2 groups were performed with the unpaired, 2-tailed Student t test, and multiple group comparisons at different time points were performed by 1- or 2-way analysis of variance (ANOVA). A value of P ≤ .05 was considered statistically significant.