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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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Coupling to endothelial nitric oxide synthasea, Western blot for HUVEC lysates probed with anti-Piezo1 antibody after transfection with Piezo1 siRNA P1.si.1 (on the left) or the control siRNA sc.si. (on the right). Prior to collection of cell lysates, HUVECs were treated with 30 ng.mL−1 VEGF (+) or no VEGF (-) for 10 min. The lysate was probed with anti-Piezo1 antibody, antibody to phosphorylated S1177 in eNOS, anti-β-actin antibody, and antibody to total eNOS protein. Positions of the expected proteins are indicated by the text on the right. The non-specific band at 250 kDa in the anti-Piezo1 blot is highlighted with *, as in Extended Data Figure 2a, b. b, Quantitative data for the down-regulation of total eNOS after transfection of HUVECs with P1.si.1 (n=6). c, Fold-change in S1177 eNOS phosphorylation (p-eNOS) evoked by VEGF (30 ng.mL−1) in HUVECs transfected with control siRNA (sc.si). or Piezo1 siRNA (P1.si.1) (n=3 each). The grey dashed line highlights 1-fold (i.e. no change). d, Western blot for VEGF (30 ng.mL−1) evoked S1177 eNOS phosphorylation (arrow) in aorta. Aorta was dissected from Piezo1+/+ or Piezo1+/− litter-mates and allowed to equilibrate at 37 oC in culture medium without shear stress for 3 h. Aorta was then exposed to VEGF (30 ng.mL−1) (+VEGF) or not (-VEGF) for 10 min, after which lysates were generated. Proteins were probed with antibody to phosphorylation at S1177 in eNOS. The band labeled with ** was not included in the analysis. The blot was also probed with anti-β-actin antibody to test for equal protein loading. e, Mean data for the type of experiment exemplified in (d) (n=5 for each genotype) and presented as in (c). f, Western blotting for HUVEC lysates after transfection with control siRNA (sc.si.) or eNOS siRNAs. The blot was probed with anti-eNOS (total) antibody. g, HUVEC migration to VEGF after incubation with vehicle control, 0.3 mM L-NMMA for 0.5 h, or 48 h after transfection with sc.si. or one of three siRNAs targeted to eNOS (n=3 each; each paired to its own control). h, Data interpretation. Error bars are s.e.m.
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Figure 11: Coupling to endothelial nitric oxide synthasea, Western blot for HUVEC lysates probed with anti-Piezo1 antibody after transfection with Piezo1 siRNA P1.si.1 (on the left) or the control siRNA sc.si. (on the right). Prior to collection of cell lysates, HUVECs were treated with 30 ng.mL−1 VEGF (+) or no VEGF (-) for 10 min. The lysate was probed with anti-Piezo1 antibody, antibody to phosphorylated S1177 in eNOS, anti-β-actin antibody, and antibody to total eNOS protein. Positions of the expected proteins are indicated by the text on the right. The non-specific band at 250 kDa in the anti-Piezo1 blot is highlighted with *, as in Extended Data Figure 2a, b. b, Quantitative data for the down-regulation of total eNOS after transfection of HUVECs with P1.si.1 (n=6). c, Fold-change in S1177 eNOS phosphorylation (p-eNOS) evoked by VEGF (30 ng.mL−1) in HUVECs transfected with control siRNA (sc.si). or Piezo1 siRNA (P1.si.1) (n=3 each). The grey dashed line highlights 1-fold (i.e. no change). d, Western blot for VEGF (30 ng.mL−1) evoked S1177 eNOS phosphorylation (arrow) in aorta. Aorta was dissected from Piezo1+/+ or Piezo1+/− litter-mates and allowed to equilibrate at 37 oC in culture medium without shear stress for 3 h. Aorta was then exposed to VEGF (30 ng.mL−1) (+VEGF) or not (-VEGF) for 10 min, after which lysates were generated. Proteins were probed with antibody to phosphorylation at S1177 in eNOS. The band labeled with ** was not included in the analysis. The blot was also probed with anti-β-actin antibody to test for equal protein loading. e, Mean data for the type of experiment exemplified in (d) (n=5 for each genotype) and presented as in (c). f, Western blotting for HUVEC lysates after transfection with control siRNA (sc.si.) or eNOS siRNAs. The blot was probed with anti-eNOS (total) antibody. g, HUVEC migration to VEGF after incubation with vehicle control, 0.3 mM L-NMMA for 0.5 h, or 48 h after transfection with sc.si. or one of three siRNAs targeted to eNOS (n=3 each; each paired to its own control). h, Data interpretation. Error bars are s.e.m.

Mentions: We next sought insight into downstream mechanisms. Account was taken of the fact that Piezo1 channel activity was stimulated by shear stress but also important for endothelial cell migration in the absence of shear stress. In nine membrane-patch recordings we had observed occasional 25-pS channel openings in the absence of mechanical strain, consistent with low-frequency Piezo1 channel activity without exogenous force. Therefore unbiased insight into downstream pathways was sought through titanium dioxide-trapping coupled with mass spectrometry to identify regulated proteins affected by Piezo1 depletion in static and shear stress conditions. Linked to Piezo1 under both conditions was endothelial nitric oxide synthase (eNOS) (Table S1), a protein with major roles in vascular biology24. Follow-up experiments confirmed reduction in total eNOS but more strikingly revealed abolition of VEGF-evoked phosphorylation of eNOS at serine 1177, a key enhancer of eNOS activity24, in static HUVECs depleted of Piezo1 and Piezo1+/− aorta in the absence of flow (Extended Data Fig. 7a-e). Consistent with functional relevance of coupling to eNOS, endothelial cell migration was similarly suppressed by Piezo1 depletion, eNOS depletion, and NOS inhibition (Extended Data Figures 2e and 7f, g). The data suggest that in the absence of shear stress Piezo1 activity drives endothelial cell migration through eNOS (Extended Data Fig. 7h).


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)

Coupling to endothelial nitric oxide synthasea, Western blot for HUVEC lysates probed with anti-Piezo1 antibody after transfection with Piezo1 siRNA P1.si.1 (on the left) or the control siRNA sc.si. (on the right). Prior to collection of cell lysates, HUVECs were treated with 30 ng.mL−1 VEGF (+) or no VEGF (-) for 10 min. The lysate was probed with anti-Piezo1 antibody, antibody to phosphorylated S1177 in eNOS, anti-β-actin antibody, and antibody to total eNOS protein. Positions of the expected proteins are indicated by the text on the right. The non-specific band at 250 kDa in the anti-Piezo1 blot is highlighted with *, as in Extended Data Figure 2a, b. b, Quantitative data for the down-regulation of total eNOS after transfection of HUVECs with P1.si.1 (n=6). c, Fold-change in S1177 eNOS phosphorylation (p-eNOS) evoked by VEGF (30 ng.mL−1) in HUVECs transfected with control siRNA (sc.si). or Piezo1 siRNA (P1.si.1) (n=3 each). The grey dashed line highlights 1-fold (i.e. no change). d, Western blot for VEGF (30 ng.mL−1) evoked S1177 eNOS phosphorylation (arrow) in aorta. Aorta was dissected from Piezo1+/+ or Piezo1+/− litter-mates and allowed to equilibrate at 37 oC in culture medium without shear stress for 3 h. Aorta was then exposed to VEGF (30 ng.mL−1) (+VEGF) or not (-VEGF) for 10 min, after which lysates were generated. Proteins were probed with antibody to phosphorylation at S1177 in eNOS. The band labeled with ** was not included in the analysis. The blot was also probed with anti-β-actin antibody to test for equal protein loading. e, Mean data for the type of experiment exemplified in (d) (n=5 for each genotype) and presented as in (c). f, Western blotting for HUVEC lysates after transfection with control siRNA (sc.si.) or eNOS siRNAs. The blot was probed with anti-eNOS (total) antibody. g, HUVEC migration to VEGF after incubation with vehicle control, 0.3 mM L-NMMA for 0.5 h, or 48 h after transfection with sc.si. or one of three siRNAs targeted to eNOS (n=3 each; each paired to its own control). h, Data interpretation. Error bars are s.e.m.
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Figure 11: Coupling to endothelial nitric oxide synthasea, Western blot for HUVEC lysates probed with anti-Piezo1 antibody after transfection with Piezo1 siRNA P1.si.1 (on the left) or the control siRNA sc.si. (on the right). Prior to collection of cell lysates, HUVECs were treated with 30 ng.mL−1 VEGF (+) or no VEGF (-) for 10 min. The lysate was probed with anti-Piezo1 antibody, antibody to phosphorylated S1177 in eNOS, anti-β-actin antibody, and antibody to total eNOS protein. Positions of the expected proteins are indicated by the text on the right. The non-specific band at 250 kDa in the anti-Piezo1 blot is highlighted with *, as in Extended Data Figure 2a, b. b, Quantitative data for the down-regulation of total eNOS after transfection of HUVECs with P1.si.1 (n=6). c, Fold-change in S1177 eNOS phosphorylation (p-eNOS) evoked by VEGF (30 ng.mL−1) in HUVECs transfected with control siRNA (sc.si). or Piezo1 siRNA (P1.si.1) (n=3 each). The grey dashed line highlights 1-fold (i.e. no change). d, Western blot for VEGF (30 ng.mL−1) evoked S1177 eNOS phosphorylation (arrow) in aorta. Aorta was dissected from Piezo1+/+ or Piezo1+/− litter-mates and allowed to equilibrate at 37 oC in culture medium without shear stress for 3 h. Aorta was then exposed to VEGF (30 ng.mL−1) (+VEGF) or not (-VEGF) for 10 min, after which lysates were generated. Proteins were probed with antibody to phosphorylation at S1177 in eNOS. The band labeled with ** was not included in the analysis. The blot was also probed with anti-β-actin antibody to test for equal protein loading. e, Mean data for the type of experiment exemplified in (d) (n=5 for each genotype) and presented as in (c). f, Western blotting for HUVEC lysates after transfection with control siRNA (sc.si.) or eNOS siRNAs. The blot was probed with anti-eNOS (total) antibody. g, HUVEC migration to VEGF after incubation with vehicle control, 0.3 mM L-NMMA for 0.5 h, or 48 h after transfection with sc.si. or one of three siRNAs targeted to eNOS (n=3 each; each paired to its own control). h, Data interpretation. Error bars are s.e.m.
Mentions: We next sought insight into downstream mechanisms. Account was taken of the fact that Piezo1 channel activity was stimulated by shear stress but also important for endothelial cell migration in the absence of shear stress. In nine membrane-patch recordings we had observed occasional 25-pS channel openings in the absence of mechanical strain, consistent with low-frequency Piezo1 channel activity without exogenous force. Therefore unbiased insight into downstream pathways was sought through titanium dioxide-trapping coupled with mass spectrometry to identify regulated proteins affected by Piezo1 depletion in static and shear stress conditions. Linked to Piezo1 under both conditions was endothelial nitric oxide synthase (eNOS) (Table S1), a protein with major roles in vascular biology24. Follow-up experiments confirmed reduction in total eNOS but more strikingly revealed abolition of VEGF-evoked phosphorylation of eNOS at serine 1177, a key enhancer of eNOS activity24, in static HUVECs depleted of Piezo1 and Piezo1+/− aorta in the absence of flow (Extended Data Fig. 7a-e). Consistent with functional relevance of coupling to eNOS, endothelial cell migration was similarly suppressed by Piezo1 depletion, eNOS depletion, and NOS inhibition (Extended Data Figures 2e and 7f, g). The data suggest that in the absence of shear stress Piezo1 activity drives endothelial cell migration through eNOS (Extended Data Fig. 7h).

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