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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 coupling to calpaina, Calpain activity indicated as absorbance (abs.) in embryos at E9.5 (n=3 Piezo1+/+, n=3 Piezo1−/−) and E10.5 (n=3 Piezo1+/+, n=3 Piezo1−/−). b, Calpain activity in HUVECs without or with shear stress (orbital shaker) for 15 min (n=3). GsMTx4 (2.5 μM). c, HUVEC alignment analysis as in Extended Data Fig. 6d, e. Test conditions: nominally Ca2+-free Krebs solution (0 Ca2+) (n=3); 3 μM PD150606 (calpain inhibitor) (n=3); 20 μM PD151746 (calpain inhibitor) (n=3); 20 μM PD145305 (negative control) (n=3); 25 μM CK59 (CaMKII inhibitor) (n=3); and 6 μM CN585 (calcineurin inhibitor) (n=3). d, Data interpretation. Error bars are s.e.m.
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Figure 4: Piezo1 coupling to calpaina, Calpain activity indicated as absorbance (abs.) in embryos at E9.5 (n=3 Piezo1+/+, n=3 Piezo1−/−) and E10.5 (n=3 Piezo1+/+, n=3 Piezo1−/−). b, Calpain activity in HUVECs without or with shear stress (orbital shaker) for 15 min (n=3). GsMTx4 (2.5 μM). c, HUVEC alignment analysis as in Extended Data Fig. 6d, e. Test conditions: nominally Ca2+-free Krebs solution (0 Ca2+) (n=3); 3 μM PD150606 (calpain inhibitor) (n=3); 20 μM PD151746 (calpain inhibitor) (n=3); 20 μM PD145305 (negative control) (n=3); 25 μM CK59 (CaMKII inhibitor) (n=3); and 6 μM CN585 (calcineurin inhibitor) (n=3). d, Data interpretation. Error bars are s.e.m.

Mentions: Nitric oxide and eNOS played no role in endothelial cell alignment to shear stress (Extended Data Fig. 8a) but in silico pathway analysis of proteomic data from endothelial cells under shear stress highlighted clusters of proteins from actin cytoskeleton (14 proteins, P=0.018) and focal adhesions (16 proteins, P=0.002). Relevance of these proteins was also indicated by denser actin structure in Piezo1-depleted cells (Extended Data Fig. 6c) and accumulation of Piezo1-GFP at the leading apical lamellipodia (Extended Data Fig. 6a, b) where focal adhesion turn-over becomes important as the endothelial cell adjusts to achieve alignment22. Moreover, detailed inspection of HUVEC and embryo proteomic data showed significant effects on calpain-2 and many of its known substrates (Extended Data Fig. 8b) (Table S2) which are important in the structure of actin cytoskeleton and focal adhesions25. We hypothesized, therefore, that a calpain-2 system is co-regulated with Piezo1 because it is integrated as a downstream mechanism. The hypothesis is consistent with calpain-2 as a Ca2+-activated proteolytic enzyme, previous association of calpain with Piezo126 and suggested roles of calpains in focal adhesion turn-over25 and endothelial cell alignment to shear stress27. Moreover, disruption of a critical regulatory protein of calpain-2 (calpain small subunit 1) disturbs vascular development in the yolk sac at E10.528. Consistent with this calpain hypothesis, calpain activity was significantly less in Piezo1−/− compared with Piezo1+/+ embryos (Fig 4a), increase in calpain activity in response to shear stress was abolished by GsMTx4 (Fig 4b), and Piezo1-GFP localized to dissolving focal adhesions at the trailing edge of the cell as shear stress was applied (Extended Data Fig. 8c). Importance of Piezo1-mediated Ca2+-entry and downstream Ca2+-activation of calpain-2 was further indicated by sensitivity of alignment to the absence of extracellular Ca2+ and presence of calpain inhibitors (Fig 4c). Inhibitors of two other Ca2+ activated mechanisms, calcineurin and Ca2+/calmodulin-dependent protein kinase II (CaMKII), had no effect (Fig 4c). The data suggest importance of calpain activation in coupling shear stress-enhanced Ca2+ entry through Piezo1 channels to endothelial cell organization and alignment via proteolytic cleavage of actin cytoskeletal and focal adhesion proteins (Fig 4d).


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 coupling to calpaina, Calpain activity indicated as absorbance (abs.) in embryos at E9.5 (n=3 Piezo1+/+, n=3 Piezo1−/−) and E10.5 (n=3 Piezo1+/+, n=3 Piezo1−/−). b, Calpain activity in HUVECs without or with shear stress (orbital shaker) for 15 min (n=3). GsMTx4 (2.5 μM). c, HUVEC alignment analysis as in Extended Data Fig. 6d, e. Test conditions: nominally Ca2+-free Krebs solution (0 Ca2+) (n=3); 3 μM PD150606 (calpain inhibitor) (n=3); 20 μM PD151746 (calpain inhibitor) (n=3); 20 μM PD145305 (negative control) (n=3); 25 μM CK59 (CaMKII inhibitor) (n=3); and 6 μM CN585 (calcineurin inhibitor) (n=3). d, Data interpretation. Error bars are s.e.m.
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Figure 4: Piezo1 coupling to calpaina, Calpain activity indicated as absorbance (abs.) in embryos at E9.5 (n=3 Piezo1+/+, n=3 Piezo1−/−) and E10.5 (n=3 Piezo1+/+, n=3 Piezo1−/−). b, Calpain activity in HUVECs without or with shear stress (orbital shaker) for 15 min (n=3). GsMTx4 (2.5 μM). c, HUVEC alignment analysis as in Extended Data Fig. 6d, e. Test conditions: nominally Ca2+-free Krebs solution (0 Ca2+) (n=3); 3 μM PD150606 (calpain inhibitor) (n=3); 20 μM PD151746 (calpain inhibitor) (n=3); 20 μM PD145305 (negative control) (n=3); 25 μM CK59 (CaMKII inhibitor) (n=3); and 6 μM CN585 (calcineurin inhibitor) (n=3). d, Data interpretation. Error bars are s.e.m.
Mentions: Nitric oxide and eNOS played no role in endothelial cell alignment to shear stress (Extended Data Fig. 8a) but in silico pathway analysis of proteomic data from endothelial cells under shear stress highlighted clusters of proteins from actin cytoskeleton (14 proteins, P=0.018) and focal adhesions (16 proteins, P=0.002). Relevance of these proteins was also indicated by denser actin structure in Piezo1-depleted cells (Extended Data Fig. 6c) and accumulation of Piezo1-GFP at the leading apical lamellipodia (Extended Data Fig. 6a, b) where focal adhesion turn-over becomes important as the endothelial cell adjusts to achieve alignment22. Moreover, detailed inspection of HUVEC and embryo proteomic data showed significant effects on calpain-2 and many of its known substrates (Extended Data Fig. 8b) (Table S2) which are important in the structure of actin cytoskeleton and focal adhesions25. We hypothesized, therefore, that a calpain-2 system is co-regulated with Piezo1 because it is integrated as a downstream mechanism. The hypothesis is consistent with calpain-2 as a Ca2+-activated proteolytic enzyme, previous association of calpain with Piezo126 and suggested roles of calpains in focal adhesion turn-over25 and endothelial cell alignment to shear stress27. Moreover, disruption of a critical regulatory protein of calpain-2 (calpain small subunit 1) disturbs vascular development in the yolk sac at E10.528. Consistent with this calpain hypothesis, calpain activity was significantly less in Piezo1−/− compared with Piezo1+/+ embryos (Fig 4a), increase in calpain activity in response to shear stress was abolished by GsMTx4 (Fig 4b), and Piezo1-GFP localized to dissolving focal adhesions at the trailing edge of the cell as shear stress was applied (Extended Data Fig. 8c). Importance of Piezo1-mediated Ca2+-entry and downstream Ca2+-activation of calpain-2 was further indicated by sensitivity of alignment to the absence of extracellular Ca2+ and presence of calpain inhibitors (Fig 4c). Inhibitors of two other Ca2+ activated mechanisms, calcineurin and Ca2+/calmodulin-dependent protein kinase II (CaMKII), had no effect (Fig 4c). The data suggest importance of calpain activation in coupling shear stress-enhanced Ca2+ entry through Piezo1 channels to endothelial cell organization and alignment via proteolytic cleavage of actin cytoskeletal and focal adhesion proteins (Fig 4d).

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