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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 mechanically-activated single channels in HUVECsa, Example single channel currents in a cell-attached patch at three voltages without subtraction of holding current. Application of −15 mmHg pressure steps to the patch pipette evoked open channel unitary currents that summated to two levels marked as O1 and O2. Closed channel current is indicated by C. b, Mean amplitudes of unitary events as exemplified in (a) and fitted with a straight line (3 patches for −50, −30 and −50 mV; 1 patch for +30 mV). c, Paired comparisons of the percentage of patches containing channel events exemplified in (a) for cells transfected with sc.si. or P1.si.1 in two independent experiment groups (n values for each group are in parentheses). In Group 2 cell-attached patch recordings cells were exposed for 10 min to 0.4 mM EGTA to chelate contaminating Ca2+ prior to recording so that sc.si.- and P1.si.1-treated cells rounded up similarly; without this treatment (Group 1), P1.si.1 but not sc.si. cells tended to round up in response to the high-K+ bath solution used to  the membrane potential of cells in cell-attached patch recordings (the reason for this effect is unknown but it may relate to changes in cytoskeleton and adhesion as discussed in relation to Fig 4). Error bars are s.e.m.
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Figure 9: Piezo1-dependence of mechanically-activated single channels in HUVECsa, Example single channel currents in a cell-attached patch at three voltages without subtraction of holding current. Application of −15 mmHg pressure steps to the patch pipette evoked open channel unitary currents that summated to two levels marked as O1 and O2. Closed channel current is indicated by C. b, Mean amplitudes of unitary events as exemplified in (a) and fitted with a straight line (3 patches for −50, −30 and −50 mV; 1 patch for +30 mV). c, Paired comparisons of the percentage of patches containing channel events exemplified in (a) for cells transfected with sc.si. or P1.si.1 in two independent experiment groups (n values for each group are in parentheses). In Group 2 cell-attached patch recordings cells were exposed for 10 min to 0.4 mM EGTA to chelate contaminating Ca2+ prior to recording so that sc.si.- and P1.si.1-treated cells rounded up similarly; without this treatment (Group 1), P1.si.1 but not sc.si. cells tended to round up in response to the high-K+ bath solution used to the membrane potential of cells in cell-attached patch recordings (the reason for this effect is unknown but it may relate to changes in cytoskeleton and adhesion as discussed in relation to Fig 4). 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 mechanically-activated single channels in HUVECsa, Example single channel currents in a cell-attached patch at three voltages without subtraction of holding current. Application of −15 mmHg pressure steps to the patch pipette evoked open channel unitary currents that summated to two levels marked as O1 and O2. Closed channel current is indicated by C. b, Mean amplitudes of unitary events as exemplified in (a) and fitted with a straight line (3 patches for −50, −30 and −50 mV; 1 patch for +30 mV). c, Paired comparisons of the percentage of patches containing channel events exemplified in (a) for cells transfected with sc.si. or P1.si.1 in two independent experiment groups (n values for each group are in parentheses). In Group 2 cell-attached patch recordings cells were exposed for 10 min to 0.4 mM EGTA to chelate contaminating Ca2+ prior to recording so that sc.si.- and P1.si.1-treated cells rounded up similarly; without this treatment (Group 1), P1.si.1 but not sc.si. cells tended to round up in response to the high-K+ bath solution used to  the membrane potential of cells in cell-attached patch recordings (the reason for this effect is unknown but it may relate to changes in cytoskeleton and adhesion as discussed in relation to Fig 4). Error bars are s.e.m.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230887&req=5

Figure 9: Piezo1-dependence of mechanically-activated single channels in HUVECsa, Example single channel currents in a cell-attached patch at three voltages without subtraction of holding current. Application of −15 mmHg pressure steps to the patch pipette evoked open channel unitary currents that summated to two levels marked as O1 and O2. Closed channel current is indicated by C. b, Mean amplitudes of unitary events as exemplified in (a) and fitted with a straight line (3 patches for −50, −30 and −50 mV; 1 patch for +30 mV). c, Paired comparisons of the percentage of patches containing channel events exemplified in (a) for cells transfected with sc.si. or P1.si.1 in two independent experiment groups (n values for each group are in parentheses). In Group 2 cell-attached patch recordings cells were exposed for 10 min to 0.4 mM EGTA to chelate contaminating Ca2+ prior to recording so that sc.si.- and P1.si.1-treated cells rounded up similarly; without this treatment (Group 1), P1.si.1 but not sc.si. cells tended to round up in response to the high-K+ bath solution used to the membrane potential of cells in cell-attached patch recordings (the reason for this effect is unknown but it may relate to changes in cytoskeleton and adhesion as discussed in relation to Fig 4). 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