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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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Global and endothelial-specific Piezo1 modification and embryonic growth retardation in micea, Simplified diagram of the Piezo1 Knockout First (conditional) construct provided in ES cells by the KOMP Repository (http://www.komp.org). Piezo1 is indicated containing insertion of lacZ sequence flanked by flippase recognition target (FRT) sites and downstream loxP sites. Further details of the construct can be obtained at http://www.komp.org. b-c, Global modification. b, Example genotyping results with lacZ or loxP-spanning PCR primers. M indicates the DNA marker ladder. On the left are results for 6 mice analysed by the lacZ PCR primers (expected product: 225 bp). On the right are the results for the same 6 mice analysed by primers targeted to endogenous Piezo1 sequence either side of the 3′ terminal loxP site (expected products: 155 bp without the loxP site; 189 bp with the loxP site). In the gel shown, 3 mice were heterozygous for the construct (+/−), 2 homozygous (−/−), and 1 wild-type (+/+). c, Images of example sibling E10.5 embryos. The embryo on the left was Piezo1+/+ and the embryo on the right was Piezo1−/−. The scale bar is 1 mm. d-g, Endothelial-specific modification. d, Example genotyping results for two mice (mouse 1 and mouse 2) both with deletion of the lacZ insert and transmission of Tie2-cre. Controls for the absence and presence of lacZ, the loxP insert, and Tie2-cre are included. Successful deletion of the lacZ insert was confirmed by lack of β-galactosidase staining (data not shown). e, Example genotyping results for six sibling embryos analysed with PCR primers spanning the deletion predicted to result from cre recombinase activity at the loxP sites. The forward primer was 5′ of the 5′ FRT site illustrated in (a) and the reverse primer was 3′ of the 3′ loxP site. The PCR product size after deletion was expected to be 379 bp. The product was detected in embryos 2 and 6. The PCR product was not generated in embryos without the deletion because it was too long to be amplified (4208 bp). Embryos exhibiting the 379 bp product were designated “EC-del.” to indicate disruptive deletion in Piezo1 of endothelial cells (ECs). Embryos designated as wild-type (wt.) exhibited no 379 bp product and only the 155 bp loxP product (as shown for the “no loxP insert control” in d). Out of a total of 142 embryos, 57 were EC-del. f, RT-PCR products detecting Piezo1 mRNA in total RNA from sibling embryos (Piezo1 3′ PCR primers) (n=3, each in duplicate). Piezo1 mRNA was significantly depleted in embryos displaying the 379 bp product described and shown in (e). g, Images of example sibling E10.5 embryos. The embryo on the left was wild-type and the embryo on the right contained endothelial-specific Piezo1 deletion (EC-del.). Retarded growth was apparent in EC-del. embryos and none of the other embryos. The scale bar is 1 mm. Error bars are s.e.m.
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Figure 7: Global and endothelial-specific Piezo1 modification and embryonic growth retardation in micea, Simplified diagram of the Piezo1 Knockout First (conditional) construct provided in ES cells by the KOMP Repository (http://www.komp.org). Piezo1 is indicated containing insertion of lacZ sequence flanked by flippase recognition target (FRT) sites and downstream loxP sites. Further details of the construct can be obtained at http://www.komp.org. b-c, Global modification. b, Example genotyping results with lacZ or loxP-spanning PCR primers. M indicates the DNA marker ladder. On the left are results for 6 mice analysed by the lacZ PCR primers (expected product: 225 bp). On the right are the results for the same 6 mice analysed by primers targeted to endogenous Piezo1 sequence either side of the 3′ terminal loxP site (expected products: 155 bp without the loxP site; 189 bp with the loxP site). In the gel shown, 3 mice were heterozygous for the construct (+/−), 2 homozygous (−/−), and 1 wild-type (+/+). c, Images of example sibling E10.5 embryos. The embryo on the left was Piezo1+/+ and the embryo on the right was Piezo1−/−. The scale bar is 1 mm. d-g, Endothelial-specific modification. d, Example genotyping results for two mice (mouse 1 and mouse 2) both with deletion of the lacZ insert and transmission of Tie2-cre. Controls for the absence and presence of lacZ, the loxP insert, and Tie2-cre are included. Successful deletion of the lacZ insert was confirmed by lack of β-galactosidase staining (data not shown). e, Example genotyping results for six sibling embryos analysed with PCR primers spanning the deletion predicted to result from cre recombinase activity at the loxP sites. The forward primer was 5′ of the 5′ FRT site illustrated in (a) and the reverse primer was 3′ of the 3′ loxP site. The PCR product size after deletion was expected to be 379 bp. The product was detected in embryos 2 and 6. The PCR product was not generated in embryos without the deletion because it was too long to be amplified (4208 bp). Embryos exhibiting the 379 bp product were designated “EC-del.” to indicate disruptive deletion in Piezo1 of endothelial cells (ECs). Embryos designated as wild-type (wt.) exhibited no 379 bp product and only the 155 bp loxP product (as shown for the “no loxP insert control” in d). Out of a total of 142 embryos, 57 were EC-del. f, RT-PCR products detecting Piezo1 mRNA in total RNA from sibling embryos (Piezo1 3′ PCR primers) (n=3, each in duplicate). Piezo1 mRNA was significantly depleted in embryos displaying the 379 bp product described and shown in (e). g, Images of example sibling E10.5 embryos. The embryo on the left was wild-type and the embryo on the right contained endothelial-specific Piezo1 deletion (EC-del.). Retarded growth was apparent in EC-del. embryos and none of the other embryos. The scale bar is 1 mm. Error bars are s.e.m.

Mentions: Messenger RNA encoding Piezo1 was readily detected in mouse aorta and a variety of human endothelial cells (Extended Data Fig. 1). To gain insight into its significance we first investigated cultured human umbilical vein endothelial cells (HUVECs). Depletion of Piezo1 by either of two short interfering RNAs (siRNAs) strongly suppressed migration of these cells towards vascular endothelial growth factor (VEGF) (Extended Data Fig. 2a-e), a key stimulant of angiogenesis in vivo9. There was similar inhibitory effect of a spider toxin blocker of Piezo1 channels, GsMTx410, and a non-specific small-molecule blocker, ruthenium red6 (Extended Data Fig. 2f). Consistent with a relationship to endothelial cell migration, HUVEC tube formations in vitro and in vivo were suppressed by Piezo1 depletion (Extended Data Fig. 2g-j). We therefore generated mice with disrupted endogenous Piezo1 gene (Extended Data Fig. 3a, b). Piezo1+/− progeny appeared normal but Piezo1−/− was embryonic lethal (Fig 1a). Of 49 Piezo1−/− embryos the longest survival time was E16.5 and most were only E9.5-11.5, which is a critical time for vascular development9. At E10.5, growth retardation was commonly observed (Extended Data Fig. 3c). At E9.5, embryos were often normal in size but yolk sac vasculature was less prominent (Fig 1b). While endothelial cells were present in yolk sacs of Piezo1+/+ and Piezo1−/− embryos the structures were different, with greater disorganization and fewer defined large vessels in Piezo1−/− (Fig 1c). Similar observations were made at E10.5 (Fig 1d). The data suggest Piezo1 as a protein of critical importance in the control of vascular architecture and embryonic development.


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)

Global and endothelial-specific Piezo1 modification and embryonic growth retardation in micea, Simplified diagram of the Piezo1 Knockout First (conditional) construct provided in ES cells by the KOMP Repository (http://www.komp.org). Piezo1 is indicated containing insertion of lacZ sequence flanked by flippase recognition target (FRT) sites and downstream loxP sites. Further details of the construct can be obtained at http://www.komp.org. b-c, Global modification. b, Example genotyping results with lacZ or loxP-spanning PCR primers. M indicates the DNA marker ladder. On the left are results for 6 mice analysed by the lacZ PCR primers (expected product: 225 bp). On the right are the results for the same 6 mice analysed by primers targeted to endogenous Piezo1 sequence either side of the 3′ terminal loxP site (expected products: 155 bp without the loxP site; 189 bp with the loxP site). In the gel shown, 3 mice were heterozygous for the construct (+/−), 2 homozygous (−/−), and 1 wild-type (+/+). c, Images of example sibling E10.5 embryos. The embryo on the left was Piezo1+/+ and the embryo on the right was Piezo1−/−. The scale bar is 1 mm. d-g, Endothelial-specific modification. d, Example genotyping results for two mice (mouse 1 and mouse 2) both with deletion of the lacZ insert and transmission of Tie2-cre. Controls for the absence and presence of lacZ, the loxP insert, and Tie2-cre are included. Successful deletion of the lacZ insert was confirmed by lack of β-galactosidase staining (data not shown). e, Example genotyping results for six sibling embryos analysed with PCR primers spanning the deletion predicted to result from cre recombinase activity at the loxP sites. The forward primer was 5′ of the 5′ FRT site illustrated in (a) and the reverse primer was 3′ of the 3′ loxP site. The PCR product size after deletion was expected to be 379 bp. The product was detected in embryos 2 and 6. The PCR product was not generated in embryos without the deletion because it was too long to be amplified (4208 bp). Embryos exhibiting the 379 bp product were designated “EC-del.” to indicate disruptive deletion in Piezo1 of endothelial cells (ECs). Embryos designated as wild-type (wt.) exhibited no 379 bp product and only the 155 bp loxP product (as shown for the “no loxP insert control” in d). Out of a total of 142 embryos, 57 were EC-del. f, RT-PCR products detecting Piezo1 mRNA in total RNA from sibling embryos (Piezo1 3′ PCR primers) (n=3, each in duplicate). Piezo1 mRNA was significantly depleted in embryos displaying the 379 bp product described and shown in (e). g, Images of example sibling E10.5 embryos. The embryo on the left was wild-type and the embryo on the right contained endothelial-specific Piezo1 deletion (EC-del.). Retarded growth was apparent in EC-del. embryos and none of the other embryos. The scale bar is 1 mm. Error bars are s.e.m.
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Related In: Results  -  Collection

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Figure 7: Global and endothelial-specific Piezo1 modification and embryonic growth retardation in micea, Simplified diagram of the Piezo1 Knockout First (conditional) construct provided in ES cells by the KOMP Repository (http://www.komp.org). Piezo1 is indicated containing insertion of lacZ sequence flanked by flippase recognition target (FRT) sites and downstream loxP sites. Further details of the construct can be obtained at http://www.komp.org. b-c, Global modification. b, Example genotyping results with lacZ or loxP-spanning PCR primers. M indicates the DNA marker ladder. On the left are results for 6 mice analysed by the lacZ PCR primers (expected product: 225 bp). On the right are the results for the same 6 mice analysed by primers targeted to endogenous Piezo1 sequence either side of the 3′ terminal loxP site (expected products: 155 bp without the loxP site; 189 bp with the loxP site). In the gel shown, 3 mice were heterozygous for the construct (+/−), 2 homozygous (−/−), and 1 wild-type (+/+). c, Images of example sibling E10.5 embryos. The embryo on the left was Piezo1+/+ and the embryo on the right was Piezo1−/−. The scale bar is 1 mm. d-g, Endothelial-specific modification. d, Example genotyping results for two mice (mouse 1 and mouse 2) both with deletion of the lacZ insert and transmission of Tie2-cre. Controls for the absence and presence of lacZ, the loxP insert, and Tie2-cre are included. Successful deletion of the lacZ insert was confirmed by lack of β-galactosidase staining (data not shown). e, Example genotyping results for six sibling embryos analysed with PCR primers spanning the deletion predicted to result from cre recombinase activity at the loxP sites. The forward primer was 5′ of the 5′ FRT site illustrated in (a) and the reverse primer was 3′ of the 3′ loxP site. The PCR product size after deletion was expected to be 379 bp. The product was detected in embryos 2 and 6. The PCR product was not generated in embryos without the deletion because it was too long to be amplified (4208 bp). Embryos exhibiting the 379 bp product were designated “EC-del.” to indicate disruptive deletion in Piezo1 of endothelial cells (ECs). Embryos designated as wild-type (wt.) exhibited no 379 bp product and only the 155 bp loxP product (as shown for the “no loxP insert control” in d). Out of a total of 142 embryos, 57 were EC-del. f, RT-PCR products detecting Piezo1 mRNA in total RNA from sibling embryos (Piezo1 3′ PCR primers) (n=3, each in duplicate). Piezo1 mRNA was significantly depleted in embryos displaying the 379 bp product described and shown in (e). g, Images of example sibling E10.5 embryos. The embryo on the left was wild-type and the embryo on the right contained endothelial-specific Piezo1 deletion (EC-del.). Retarded growth was apparent in EC-del. embryos and none of the other embryos. The scale bar is 1 mm. Error bars are s.e.m.
Mentions: Messenger RNA encoding Piezo1 was readily detected in mouse aorta and a variety of human endothelial cells (Extended Data Fig. 1). To gain insight into its significance we first investigated cultured human umbilical vein endothelial cells (HUVECs). Depletion of Piezo1 by either of two short interfering RNAs (siRNAs) strongly suppressed migration of these cells towards vascular endothelial growth factor (VEGF) (Extended Data Fig. 2a-e), a key stimulant of angiogenesis in vivo9. There was similar inhibitory effect of a spider toxin blocker of Piezo1 channels, GsMTx410, and a non-specific small-molecule blocker, ruthenium red6 (Extended Data Fig. 2f). Consistent with a relationship to endothelial cell migration, HUVEC tube formations in vitro and in vivo were suppressed by Piezo1 depletion (Extended Data Fig. 2g-j). We therefore generated mice with disrupted endogenous Piezo1 gene (Extended Data Fig. 3a, b). Piezo1+/− progeny appeared normal but Piezo1−/− was embryonic lethal (Fig 1a). Of 49 Piezo1−/− embryos the longest survival time was E16.5 and most were only E9.5-11.5, which is a critical time for vascular development9. At E10.5, growth retardation was commonly observed (Extended Data Fig. 3c). At E9.5, embryos were often normal in size but yolk sac vasculature was less prominent (Fig 1b). While endothelial cells were present in yolk sacs of Piezo1+/+ and Piezo1−/− embryos the structures were different, with greater disorganization and fewer defined large vessels in Piezo1−/− (Fig 1c). Similar observations were made at E10.5 (Fig 1d). The data suggest Piezo1 as a protein of critical importance in the control of vascular architecture and embryonic development.

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