Figure 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).

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).

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