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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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Shear stress-evoked redistribution of Piezo1 and the role of Piezo1 in alignment of endothelial cells to the direction of shear stressThe application and direction of shear stress is indicated by open arrows and the cells were HUVECs. a, The left-hand image is of Piezo1-GFP in a single cell with a box indicating the region expanded in the middle and right-hand images after 0 and 50 min 15 dyn.cm−2 in the microfluidic chamber. In the left image i indicates the part of the cell that became trailing after application of shear stress and ii that which became leading. Scale bars, 10 μm. b, Analysis of experiments of the type shown in (a) (n=8 per data point except for n=7 at 50 min) where i and ii indicate the trailing and leading edges of the cell as shown in (a). c, Example cells after 24 h shear stress caused by the orbital shaker. Rhodamine phalloidin labeled F-actin (red) and DAPI labeled cell nuclei (blue). A paired comparison was made of cells transfected with control siRNA (sc.si.) or Piezo1 siRNA (P1.si.1). Scale bars, 50 μm. d, Example orientation analysis for pairs of images of the type shown in (c). e, As for (d) but normalized mean data for the frequency (number of angles) at the mode in experiments comparing mock with P1.si.1 transfected cells (n=5 each) and 2.5 μM GsMTx4 with its vehicle control (n=4 each). There is also comparison of cells transfected with sc.si. or P1.si.1 after 15 h of 15 dyn.cm−2 in the microfluidic chamber (n=3). Error bars are s.e.m.
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Figure 10: Shear stress-evoked redistribution of Piezo1 and the role of Piezo1 in alignment of endothelial cells to the direction of shear stressThe application and direction of shear stress is indicated by open arrows and the cells were HUVECs. a, The left-hand image is of Piezo1-GFP in a single cell with a box indicating the region expanded in the middle and right-hand images after 0 and 50 min 15 dyn.cm−2 in the microfluidic chamber. In the left image i indicates the part of the cell that became trailing after application of shear stress and ii that which became leading. Scale bars, 10 μm. b, Analysis of experiments of the type shown in (a) (n=8 per data point except for n=7 at 50 min) where i and ii indicate the trailing and leading edges of the cell as shown in (a). c, Example cells after 24 h shear stress caused by the orbital shaker. Rhodamine phalloidin labeled F-actin (red) and DAPI labeled cell nuclei (blue). A paired comparison was made of cells transfected with control siRNA (sc.si.) or Piezo1 siRNA (P1.si.1). Scale bars, 50 μm. d, Example orientation analysis for pairs of images of the type shown in (c). e, As for (d) but normalized mean data for the frequency (number of angles) at the mode in experiments comparing mock with P1.si.1 transfected cells (n=5 each) and 2.5 μM GsMTx4 with its vehicle control (n=4 each). There is also comparison of cells transfected with sc.si. or P1.si.1 after 15 h of 15 dyn.cm−2 in the microfluidic chamber (n=3). Error bars are s.e.m.

Mentions: To shed light on the functional significance of shear stress-activated Piezo1 channels we first tracked the sub cellular localization of Piezo1 tagged with green fluorescent protein (GFP). In static conditions it was broadly distributed but in response to shear stress there was accumulation at the leading apical lamellipodia (Extended Data Fig. 6a, b). Such apical processes22 are characteristic of early-stage alignment of endothelial cells in the direction of shear stress, a process occurring physiologically in blood vessels2,23. We therefore measured alignment of HUVECs and found it to be suppressed by Piezo1 depletion or GsMTx4 (Extended Data Fig. 6c-e). Endothelial cells isolated from Piezo1−/− embryos also showed less alignment (Fig 3a, b). To investigate alignment in the adult animal we took advantage of haploinsufficiency in Piezo1+/− mice (Fig 3c). Imaging of CD31-positive cells in arteries of these mice at 6-8 weeks revealed striking difference in the organization of the endothelial cells: a cobblestone-like appearance in Piezo1+/− and linear appearance in the direction of flow in Piezo1+/+ litter-mate controls (Fig 3d, e). The data suggest Piezo1 channels as shear stress sensors that promote endothelial cell organization and alignment in the direction of flow.


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)

Shear stress-evoked redistribution of Piezo1 and the role of Piezo1 in alignment of endothelial cells to the direction of shear stressThe application and direction of shear stress is indicated by open arrows and the cells were HUVECs. a, The left-hand image is of Piezo1-GFP in a single cell with a box indicating the region expanded in the middle and right-hand images after 0 and 50 min 15 dyn.cm−2 in the microfluidic chamber. In the left image i indicates the part of the cell that became trailing after application of shear stress and ii that which became leading. Scale bars, 10 μm. b, Analysis of experiments of the type shown in (a) (n=8 per data point except for n=7 at 50 min) where i and ii indicate the trailing and leading edges of the cell as shown in (a). c, Example cells after 24 h shear stress caused by the orbital shaker. Rhodamine phalloidin labeled F-actin (red) and DAPI labeled cell nuclei (blue). A paired comparison was made of cells transfected with control siRNA (sc.si.) or Piezo1 siRNA (P1.si.1). Scale bars, 50 μm. d, Example orientation analysis for pairs of images of the type shown in (c). e, As for (d) but normalized mean data for the frequency (number of angles) at the mode in experiments comparing mock with P1.si.1 transfected cells (n=5 each) and 2.5 μM GsMTx4 with its vehicle control (n=4 each). There is also comparison of cells transfected with sc.si. or P1.si.1 after 15 h of 15 dyn.cm−2 in the microfluidic chamber (n=3). Error bars are s.e.m.
© Copyright Policy
Related In: Results  -  Collection

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Figure 10: Shear stress-evoked redistribution of Piezo1 and the role of Piezo1 in alignment of endothelial cells to the direction of shear stressThe application and direction of shear stress is indicated by open arrows and the cells were HUVECs. a, The left-hand image is of Piezo1-GFP in a single cell with a box indicating the region expanded in the middle and right-hand images after 0 and 50 min 15 dyn.cm−2 in the microfluidic chamber. In the left image i indicates the part of the cell that became trailing after application of shear stress and ii that which became leading. Scale bars, 10 μm. b, Analysis of experiments of the type shown in (a) (n=8 per data point except for n=7 at 50 min) where i and ii indicate the trailing and leading edges of the cell as shown in (a). c, Example cells after 24 h shear stress caused by the orbital shaker. Rhodamine phalloidin labeled F-actin (red) and DAPI labeled cell nuclei (blue). A paired comparison was made of cells transfected with control siRNA (sc.si.) or Piezo1 siRNA (P1.si.1). Scale bars, 50 μm. d, Example orientation analysis for pairs of images of the type shown in (c). e, As for (d) but normalized mean data for the frequency (number of angles) at the mode in experiments comparing mock with P1.si.1 transfected cells (n=5 each) and 2.5 μM GsMTx4 with its vehicle control (n=4 each). There is also comparison of cells transfected with sc.si. or P1.si.1 after 15 h of 15 dyn.cm−2 in the microfluidic chamber (n=3). Error bars are s.e.m.
Mentions: To shed light on the functional significance of shear stress-activated Piezo1 channels we first tracked the sub cellular localization of Piezo1 tagged with green fluorescent protein (GFP). In static conditions it was broadly distributed but in response to shear stress there was accumulation at the leading apical lamellipodia (Extended Data Fig. 6a, b). Such apical processes22 are characteristic of early-stage alignment of endothelial cells in the direction of shear stress, a process occurring physiologically in blood vessels2,23. We therefore measured alignment of HUVECs and found it to be suppressed by Piezo1 depletion or GsMTx4 (Extended Data Fig. 6c-e). Endothelial cells isolated from Piezo1−/− embryos also showed less alignment (Fig 3a, b). To investigate alignment in the adult animal we took advantage of haploinsufficiency in Piezo1+/− mice (Fig 3c). Imaging of CD31-positive cells in arteries of these mice at 6-8 weeks revealed striking difference in the organization of the endothelial cells: a cobblestone-like appearance in Piezo1+/− and linear appearance in the direction of flow in Piezo1+/+ litter-mate controls (Fig 3d, e). The data suggest Piezo1 channels as shear stress sensors that promote endothelial cell organization and alignment in the direction of flow.

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