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Piezo1 integration of vascular architecture with physiological force.

Li J, Hou B, Tumova S, Muraki K, Bruns A, Ludlow MJ, Sedo A, Hyman AJ, McKeown L, Young RS, Yuldasheva NY, Majeed Y, Wilson LA, Rode B, Bailey MA, Kim HR, Fu Z, Carter DA, Bilton J, Imrie H, Ajuh P, Dear TN, Cubbon RM, Kearney MT, Prasad KR, Evans PC, Ainscough JF, Beech DJ - Nature (2014)

Bottom Line: Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating.Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force.The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Medicine and Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds LS2 9JT, UK [2].

ABSTRACT
The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

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Piezo1-dependence of shear stress-evoked Ca2+ events in human endothelial cells and mouse embryonic endothelial cellsa, Example intracellular Ca2+ events evoked by microfluidic shear stress in HUVECs transfected with control siRNA (sc.si.) or Piezo1.si.1 (P1.si.1). Each trace is for 1 cell. In one P1.si.1 cell, transient Ca2+ elevation remained. Such residual events may reflect insufficient Piezo1 depletion in some cells or non-Piezo1 mechanisms. b, Mean data for experiments of the type in (a) and expanded to paired comparisons of sc.si. and P1.si.1 (n=5 each), sc.si. and P1.si.2 (n=4 each), vehicle and 2.5 μM GsMTx4 (n=3 each). Data were normalized to their respective controls. c, Quantification of Piezo1 mRNA depletion (n=4 each) plotted against the inhibition of the intracellular Ca2+ elevations evoked by 20 dyn.cm−2. Three different Piezo1 siRNAs were compared with their control siRNAs. The Ca2+ data are from the experiments described in (b). Sequence details of the siRNAs are provided in Table S3. d, Mean Ca2+ signals evoked by 20 dyn.cm−2 in non-transfected HUVECs. Measurements were made in standard bath solution without the addition of an inhibitor (no inhibitor) (n=8), 10 μM gadolinium chloride (Gd3+) (n=3), or with Ca2+ omitted from the bath solution (0 Ca2+) (n=3). e, Ca2+ release evoked by 2 μM thapsigargin (TG) in the absence of extracellular Ca2+ and after transfection with sc.si. or P1.si.1 (20 wells of a 96-well plate each). f, Mean data normalized to sc.si. for experiments of the type shown in (e) and analysed for the rate of rise of the Ca2+ event evoked by TG (n=3 each). g, Similar to (b) but endothelial cells were from patient liver samples, data were not normalized, and only P1.si.1 was used (n=3, 4, 10 and 5 for shear stresses of 5, 10, 15 and 20 dyn.cm−2). h, i, Intracellular Ca2+ measurements from mouse embryonic endothelial cells in microfluidic chambers. h, Superimposition of example intracellular Ca2+ events in 2 single cells on different coverslips from Piezo1+/+ and Piezo1−/− sibling embryos. Shear stress was applied at 15 and 25 dyn.cm−2 and then 30 ng.mL−1 VEGF was introduced while maintaining shear stress at 25 dyn.cm−2. i, Mean±s.e.mean data for all VEGF-responsive cells studied as exemplified in (h) (n=6 +/+, 54 cells; n=5 −/−, 42 cells). The same data are summarized in simplified form in Fig 2a. Error bars are s.e.m.
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Figure 8: Piezo1-dependence of shear stress-evoked Ca2+ events in human endothelial cells and mouse embryonic endothelial cellsa, Example intracellular Ca2+ events evoked by microfluidic shear stress in HUVECs transfected with control siRNA (sc.si.) or Piezo1.si.1 (P1.si.1). Each trace is for 1 cell. In one P1.si.1 cell, transient Ca2+ elevation remained. Such residual events may reflect insufficient Piezo1 depletion in some cells or non-Piezo1 mechanisms. b, Mean data for experiments of the type in (a) and expanded to paired comparisons of sc.si. and P1.si.1 (n=5 each), sc.si. and P1.si.2 (n=4 each), vehicle and 2.5 μM GsMTx4 (n=3 each). Data were normalized to their respective controls. c, Quantification of Piezo1 mRNA depletion (n=4 each) plotted against the inhibition of the intracellular Ca2+ elevations evoked by 20 dyn.cm−2. Three different Piezo1 siRNAs were compared with their control siRNAs. The Ca2+ data are from the experiments described in (b). Sequence details of the siRNAs are provided in Table S3. d, Mean Ca2+ signals evoked by 20 dyn.cm−2 in non-transfected HUVECs. Measurements were made in standard bath solution without the addition of an inhibitor (no inhibitor) (n=8), 10 μM gadolinium chloride (Gd3+) (n=3), or with Ca2+ omitted from the bath solution (0 Ca2+) (n=3). e, Ca2+ release evoked by 2 μM thapsigargin (TG) in the absence of extracellular Ca2+ and after transfection with sc.si. or P1.si.1 (20 wells of a 96-well plate each). f, Mean data normalized to sc.si. for experiments of the type shown in (e) and analysed for the rate of rise of the Ca2+ event evoked by TG (n=3 each). g, Similar to (b) but endothelial cells were from patient liver samples, data were not normalized, and only P1.si.1 was used (n=3, 4, 10 and 5 for shear stresses of 5, 10, 15 and 20 dyn.cm−2). h, i, Intracellular Ca2+ measurements from mouse embryonic endothelial cells in microfluidic chambers. h, Superimposition of example intracellular Ca2+ events in 2 single cells on different coverslips from Piezo1+/+ and Piezo1−/− sibling embryos. Shear stress was applied at 15 and 25 dyn.cm−2 and then 30 ng.mL−1 VEGF was introduced while maintaining shear stress at 25 dyn.cm−2. i, Mean±s.e.mean data for all VEGF-responsive cells studied as exemplified in (h) (n=6 +/+, 54 cells; n=5 −/−, 42 cells). The same data are summarized in simplified form in Fig 2a. Error bars are s.e.m.

Mentions: Piezo1 depletion and GsMTx4 were found to suppress shear stress-evoked Ca2+ entry in HUVECs (Extended Data Fig. 4a-f). Hepatic endothelial cells from patients undergoing surgical liver resection were also investigated and had similar dependency on Piezo1 (Extended Data Fig. 4g). Moreover, Piezo1−/− embryonic endothelial cells had less shear stress-evoked Ca2+ entry (Fig 2a) (Extended Data Fig. 4h, i). Furthermore, ionic current reversibly induced by shear stress had a current-voltage relationship (I-V) that was linear and reversed near 0 mV, as expected for Piezo1 channels6, and Piezo1 depletion suppressed the current (Fig 2b-d). In cell-attached membrane patches, negative pressure used to deliver physical force evoked unitary single channel events within less than 1 s. The unitary conductance of these channels was 25.2±1.7 pS, consistent with Piezo1 channels6, and Piezo1 depletion depleted the channels (Extended Data Fig. 5). The data suggest importance of Piezo1 channels in shear stress-sensing and the associated Ca2+ entry of endothelial cells.


Piezo1 integration of vascular architecture with physiological force.

Li J, Hou B, Tumova S, Muraki K, Bruns A, Ludlow MJ, Sedo A, Hyman AJ, McKeown L, Young RS, Yuldasheva NY, Majeed Y, Wilson LA, Rode B, Bailey MA, Kim HR, Fu Z, Carter DA, Bilton J, Imrie H, Ajuh P, Dear TN, Cubbon RM, Kearney MT, Prasad KR, Evans PC, Ainscough JF, Beech DJ - Nature (2014)

Piezo1-dependence of shear stress-evoked Ca2+ events in human endothelial cells and mouse embryonic endothelial cellsa, Example intracellular Ca2+ events evoked by microfluidic shear stress in HUVECs transfected with control siRNA (sc.si.) or Piezo1.si.1 (P1.si.1). Each trace is for 1 cell. In one P1.si.1 cell, transient Ca2+ elevation remained. Such residual events may reflect insufficient Piezo1 depletion in some cells or non-Piezo1 mechanisms. b, Mean data for experiments of the type in (a) and expanded to paired comparisons of sc.si. and P1.si.1 (n=5 each), sc.si. and P1.si.2 (n=4 each), vehicle and 2.5 μM GsMTx4 (n=3 each). Data were normalized to their respective controls. c, Quantification of Piezo1 mRNA depletion (n=4 each) plotted against the inhibition of the intracellular Ca2+ elevations evoked by 20 dyn.cm−2. Three different Piezo1 siRNAs were compared with their control siRNAs. The Ca2+ data are from the experiments described in (b). Sequence details of the siRNAs are provided in Table S3. d, Mean Ca2+ signals evoked by 20 dyn.cm−2 in non-transfected HUVECs. Measurements were made in standard bath solution without the addition of an inhibitor (no inhibitor) (n=8), 10 μM gadolinium chloride (Gd3+) (n=3), or with Ca2+ omitted from the bath solution (0 Ca2+) (n=3). e, Ca2+ release evoked by 2 μM thapsigargin (TG) in the absence of extracellular Ca2+ and after transfection with sc.si. or P1.si.1 (20 wells of a 96-well plate each). f, Mean data normalized to sc.si. for experiments of the type shown in (e) and analysed for the rate of rise of the Ca2+ event evoked by TG (n=3 each). g, Similar to (b) but endothelial cells were from patient liver samples, data were not normalized, and only P1.si.1 was used (n=3, 4, 10 and 5 for shear stresses of 5, 10, 15 and 20 dyn.cm−2). h, i, Intracellular Ca2+ measurements from mouse embryonic endothelial cells in microfluidic chambers. h, Superimposition of example intracellular Ca2+ events in 2 single cells on different coverslips from Piezo1+/+ and Piezo1−/− sibling embryos. Shear stress was applied at 15 and 25 dyn.cm−2 and then 30 ng.mL−1 VEGF was introduced while maintaining shear stress at 25 dyn.cm−2. i, Mean±s.e.mean data for all VEGF-responsive cells studied as exemplified in (h) (n=6 +/+, 54 cells; n=5 −/−, 42 cells). The same data are summarized in simplified form in Fig 2a. Error bars are s.e.m.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230887&req=5

Figure 8: Piezo1-dependence of shear stress-evoked Ca2+ events in human endothelial cells and mouse embryonic endothelial cellsa, Example intracellular Ca2+ events evoked by microfluidic shear stress in HUVECs transfected with control siRNA (sc.si.) or Piezo1.si.1 (P1.si.1). Each trace is for 1 cell. In one P1.si.1 cell, transient Ca2+ elevation remained. Such residual events may reflect insufficient Piezo1 depletion in some cells or non-Piezo1 mechanisms. b, Mean data for experiments of the type in (a) and expanded to paired comparisons of sc.si. and P1.si.1 (n=5 each), sc.si. and P1.si.2 (n=4 each), vehicle and 2.5 μM GsMTx4 (n=3 each). Data were normalized to their respective controls. c, Quantification of Piezo1 mRNA depletion (n=4 each) plotted against the inhibition of the intracellular Ca2+ elevations evoked by 20 dyn.cm−2. Three different Piezo1 siRNAs were compared with their control siRNAs. The Ca2+ data are from the experiments described in (b). Sequence details of the siRNAs are provided in Table S3. d, Mean Ca2+ signals evoked by 20 dyn.cm−2 in non-transfected HUVECs. Measurements were made in standard bath solution without the addition of an inhibitor (no inhibitor) (n=8), 10 μM gadolinium chloride (Gd3+) (n=3), or with Ca2+ omitted from the bath solution (0 Ca2+) (n=3). e, Ca2+ release evoked by 2 μM thapsigargin (TG) in the absence of extracellular Ca2+ and after transfection with sc.si. or P1.si.1 (20 wells of a 96-well plate each). f, Mean data normalized to sc.si. for experiments of the type shown in (e) and analysed for the rate of rise of the Ca2+ event evoked by TG (n=3 each). g, Similar to (b) but endothelial cells were from patient liver samples, data were not normalized, and only P1.si.1 was used (n=3, 4, 10 and 5 for shear stresses of 5, 10, 15 and 20 dyn.cm−2). h, i, Intracellular Ca2+ measurements from mouse embryonic endothelial cells in microfluidic chambers. h, Superimposition of example intracellular Ca2+ events in 2 single cells on different coverslips from Piezo1+/+ and Piezo1−/− sibling embryos. Shear stress was applied at 15 and 25 dyn.cm−2 and then 30 ng.mL−1 VEGF was introduced while maintaining shear stress at 25 dyn.cm−2. i, Mean±s.e.mean data for all VEGF-responsive cells studied as exemplified in (h) (n=6 +/+, 54 cells; n=5 −/−, 42 cells). The same data are summarized in simplified form in Fig 2a. Error bars are s.e.m.
Mentions: Piezo1 depletion and GsMTx4 were found to suppress shear stress-evoked Ca2+ entry in HUVECs (Extended Data Fig. 4a-f). Hepatic endothelial cells from patients undergoing surgical liver resection were also investigated and had similar dependency on Piezo1 (Extended Data Fig. 4g). Moreover, Piezo1−/− embryonic endothelial cells had less shear stress-evoked Ca2+ entry (Fig 2a) (Extended Data Fig. 4h, i). Furthermore, ionic current reversibly induced by shear stress had a current-voltage relationship (I-V) that was linear and reversed near 0 mV, as expected for Piezo1 channels6, and Piezo1 depletion suppressed the current (Fig 2b-d). In cell-attached membrane patches, negative pressure used to deliver physical force evoked unitary single channel events within less than 1 s. The unitary conductance of these channels was 25.2±1.7 pS, consistent with Piezo1 channels6, and Piezo1 depletion depleted the channels (Extended Data Fig. 5). The data suggest importance of Piezo1 channels in shear stress-sensing and the associated Ca2+ entry of endothelial cells.

Bottom Line: Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating.Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force.The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Medicine and Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds LS2 9JT, UK [2].

ABSTRACT
The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

Show MeSH
Related in: MedlinePlus