The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic1-5. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact6-8. 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. 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 was protease activity and spatial organization of endothelial cells to the polarity of the applied force. The data suggest Piezo1 channels as pivotal integrators in vascular biology.
Background and PurposeThe mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small‐molecule agonist, but the pharmacology of these channels is otherwise limited.Experimental ApproachYoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+ and Tl+ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta.Key ResultsModification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1‐induced Ca2+‐entry with IC50s of 1.3 μM (HEK 293 cells) and 1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP‐evoked Ca2+ elevation or store‐operated Ca2+ entry in HEK 293 cells or Ca2+ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose‐dependent relaxation of aortic rings, which was mediated by an endothelium‐ and NO‐dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1.Conclusion and ImplicationsChemical antagonism of Yoda1‐evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.
Summary Endogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.
LCM was funded by a Clinical Research Training Fellowship from the Medical Research Council and by the Royal College of Obstetricians and Gynaecologists, and has received support from a Wellcome Trust Institutional Strategic Support Fund. JS was supported by the Wellcome Trust and a BHF Intermediate Research Fellowship. HJG, CW, AJH and PJW were supported by PhD Studentships from BHF, BBSRC and the Leeds Teaching Hospitals Charitable Foundation respectively. All authors declare no conflict of interest.
Piezo1 forms a mechanically activated calcium-permeable nonselective cation channel that is functionally important in many cell types. Structural data exist for C-terminal regions, but we lack information about N-terminal regions and how the entire channel interacts with the lipid bilayer. Here, we use computational approaches to predict the three-dimensional structure of the full-length Piezo1 and simulate it in an asymmetric membrane. A number of novel insights are suggested by the model: 1) Piezo1 creates a trilobed dome in the membrane that extends beyond the radius of the protein, 2) Piezo1 changes the lipid environment in its vicinity via preferential interactions with cholesterol and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) molecules, and 3) cholesterol changes the depth of the dome and PIP 2 binding preference. In vitro alteration of cholesterol concentration inhibits Piezo1 activity in a manner complementing some of our computational findings. The data suggest the importance of N-terminal regions of Piezo1 for dome structure and membrane cholesterol and PIP 2 interactions.
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions. All original data are available in an Excel file. Unique laboratory materials created in the project are available on request (D.J.B. for biological materials and R.F. for chemicals). FUNDING STATEMENTThe work was supported by research grants from Wellcome (grant number 110044/Z/15/Z) and British Heart Foundation (grant number RG/17/11/33042) and studentships from University of Leeds (for K.C.) and BBSRC (for A.J.H.). For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. AUTHOR CONTRIBUTIONS G.P., A.J.H. and J.A.K. designed and performed calcium measurement assays. K.C. and C.H.R. designed, synthesized and analysed chemicals. N.E. designed and performed myography assays. O.V.P. designed and performed PIEZO1 patch-clamp experiments and J.A.K. PIEZO2 patch-clamp experiments. N.M., M.G.R., N.B. and A.B. designed automated patch-clamp experiments. N.M. and M.G.R. performed automated patch-clamp assays. T.S.F. and L.L. bred and maintained genetically engineered mice. M.J.L. generated cell lines. G.P., N.E., N.M. and O.V.P. made the figures. G.P. orchestrated the figure designs, data analysis and data transparency. CHR generated the supplementary chemistry information. E.C.-B. made intellectual contribution and performed PIEZO2 and HeLa cell experiments with M.D. and F.B.. G.P. and D.J.B. interpreted data and wrote most of the manuscript with input from
The cytoprotective effects of prostaglandins have been utilized in the prevention of hepatitis B virus reactivation after liver transplantation. This pilot study evaluated the effects of oral prostaglandin E2 (PGE2) in chronic viral hepatitis B and C. Twenty patients with chronic hepatitis B and 20 patients with chronic hepatitis C received 4mg day-1 PGE2 for 6 months. The lymphocyte antiviral enzyme 2',5'-oligoadenylate synthetase (2',5'-OAS) and peripheral blood monocyte procoagulant activity (PCA) were measured before, during and after the treatment. Three of 20 hepatitis B and five of 20 hepatitis C patients withdrew from the study. Eight of 17 hepatitis B patients responded: in seven of these eight patients, serum alanine aminotransferase (ALT) levels normalized; loss of viral replication was sustained in all eight patients; and seroconversion from hepatitis Be antigen (HBeAg) to hepatitis Be antibody (HBeAb) positivity occurred in seven patients over the 48-week duration of this study. In 14 of the 15 hepatitis C patients, hepatitis C virus (HCV) RNA remained detectable and the serum ALT levels remained elevated. 2',5'-OAS levels and PCA values did not correlate with other markers of response to PGE2 therapy in either chronic hepatitis B or C. In summary, PGE2 was associated with sustained loss of viral replication in 47% of chronic hepatitis B patients; no beneficial effects were apparent in chronic hepatitis C.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.