Mechanically activated ion channel PIEZO1 is required for lymphatic valve formation

Abstract

PIEZO1 is a cation channel that is activated by mechanical forces such as fluid shear stress or membrane stretch. PIEZO1 loss-of-function mutations in patients are associated with congenital lymphedema with pleural effusion. However, the mechanistic link between PIEZO1 function and the development or function of the lymphatic system is currently unknown. Here, we analyzed two mouse lines lacking PIEZO1 in endothelial cells (via Tie2Cre or Lyve1Cre) and found that they exhibited pleural effusion and died postnatally. Strikingly, the number of lymphatic valves was dramatically reduced in these mice. Lymphatic valves are essential for ensuring proper circulation of lymph. Mechanical forces have been implicated in the development of lymphatic vasculature and valve formation, but the identity of mechanosensors involved is unknown. Expression of FOXC2 and NFATc1, transcription factors known to be required for lymphatic valve development, appeared normal in Tie2Cre;Piezo1^cKO mice. However, the process of protrusion in the valve leaflets, which is associated with collective cell migration, actin polymerization, and remodeling of cell-cell junctions, was impaired in Tie2Cre;Piezo1^cKO mice. Consistent with these genetic findings, activation of PIEZO1 by Yoda1 in cultured lymphatic endothelial cells induced active remodeling of actomyosin and VE-cadherin⁺ cell-cell adhesion sites. Our analysis provides evidence that mechanically activated ion channel PIEZO1 is a key regulator of lymphatic valve formation.

Keywords: PIEZO1; ion channel; lymphatic system; mechanotransduction; valve formation.

Fig. 1.

Postnatal lethality, pleural effusion in endothelial-specific Piezo1 cKO mice. (A) Survival rate of WT (n = 14), Het (Piezo1^−/+ or Tie2Cre;Piezo1^+/fl) (n = 13), or Tie2Cre;Piezo1^cKO (n = 14) newborn mice. (B) Chylous pleural effusion (red arrow) observed in 1-wk-old Tie2Cre;Piezo1^cKO, but not in WT littermate. (Scale bar: 5 mm.) (C) Amount of liquid in the chest cavity at P9. Mean ± SEM (n = 6 for WT, n = 5 for Tie2Cre;Piezo1^cKO mice). *P < 0.05, unpaired t test. (D) Survival rate of WT (n = 13) or Lyve1Cre;Piezo1^cKO (n = 15) newborn mice. (E) Chylous pleural effusion (red arrows) observed in 1-wk-old Lyve1Cre;Piezo1^cKO mice, but not in WT littermate. (Scale bar: 5 mm.) (F) Amount of liquid in the chest cavity in 1-wk-old mice. Mean ± SEM (n = 16 for WT, n = 8 for Lyve1Cre;Piezo1^cKO mice). *P < 0.05, unpaired t test.

Fig. 2.

Reduced number of lymphatic valves in endothelial-specific Piezo1 cKO mice. (A and C) Prox1 and integrin α9 immunostaining in P4 mesentery of WT, Tie2Cre;Piezo1^cKO (A) or Lyve1Cre;Piezo1^cKO mice (C). Arrowheads indicate lymphatic valves marked by Prox1 and integrin α9. (Scale bar: 100 μm.) (B and D) Number of lymphatic valves per area in P4 mesentery. Mean ± SEM n = 4 for both WT and Tie2Cre;Piezo1^cKO samples, **P < 0.01 (B). n = 6 for WT, n = 4 for Lyve1Cre;Piezo1^cKO samples, ****P < 0.0001 (D), unpaired t test.

Fig. 3.

Impaired lymphatic valve protrusion in Tie2Cre;Piezo1^cKO mice. (A) E18.5 mesentery with Prox1 staining. (Scale bar: 100 μm.) (B) Number of VFRs (containing n ≥ 10 Prox1^high nuclei) per area in E18.5 mesentery. Mean ± SEM (n = 4 for WT, n = 5 for Tie2Cre;Piezo1^cKO embryos). P = 0.35, unpaired t test. (C) Stages of lymphatic valve formation. Stage 2: there are n < 5 reoriented Prox1^high nuclei and the vessel is not constricted. Stage 2.5: n ≥ 5 Prox1^high nuclei are reoriented but the vessel is not constricted. Stages 3 and 4: n ≥ 5 Prox1^high nuclei are reoriented and the vessel is constricted. (D) Percentage of each stage among VFRs in E18.5 mesentery. n = 17 VFRs from four embryos per genotype. *P < 0.05, χ² test. (E) Representative images of VFRs in E18.5 mesentery. Yellow lines (Top, side view) show where images were optically cross-sectioned (Middle) and schematic illustration of distribution of Prox1^high nucleus (green), VE-cadherin (red), and F-actin (blue) signals (Bottom). Dotted lines demarcate lymphatic vessels. (Scale bar: 100 μm.) (F–H) Percentage of VFRs in E18.5 mesentery in which several Prox1^high nuclei (F), VE-cadherin (G), or F-actin (H) signal was located in the more central regions than Prox1^high nuclei on the vessel wall. Right shows examples of patterns of each signal. n = 10 images from four WT embryos, n = 14 images from four Tie2Cre;Piezo1^cKO embryos. *P < 0.05, χ² test.

Fig. 4.

Expression of transcription factors involved in initiation of valve development in Tie2Cre;Piezo1^cKO mice. (A and B) FOXC2 (A) or NFATc1 (B) with Prox1 staining in E17.5 mesentery of WT or Tie2Cre;Piezo1^cKO embryos. Insets show magnified images. FOXC2: n = 8 for WT, n = 7 for Tie2Cre;Piezo1^cKO embryos. NFATc1: n = 7 for WT, n = 6 for Tie2Cre;Piezo1^cKO embryos. (Scale bar: 100 μm.)

Fig. 5.

PIEZO1 activation-induced changes in actomyosin and VE-cadherin⁺ cell–cell adhesion in cultured LECs. (A) Staining of F-actin, pMLC2, and VE-cadherin in LECs treated with 1.5 μM Yoda1 for 0, 1, or 16 h. (Scale bar: 50 μm.) (B and C) Staining of F-actin, pMLC2, and VE-cadherin in LECs treated with control (B) or PIEZO1 siRNA (C) before Yoda1 administration. n = 2 experiments.