Abstract
Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis1. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses2. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures3,4,5. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature.
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Data availability
The datasets generated during and/or analysed during this study are either included within the manuscript or are available from the corresponding author on reasonable request. Source Data for Figs. 1–4 and Extended Data Figs. 2–11 are provided with the paper. Gel source data can be found in Supplementary Fig. 1.
Change history
03 May 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41586-022-04815-w
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Acknowledgements
This work was supported in part by grants from the Wellcome Trust (Senior Research Fellowship to E.T.), BHF (PG/16/29/32128 to E.T.), John Fell Fund (to E.T.) the BHF Centre of Excellence, Oxford (RE/13/1/30181), Cancer Research UK and the UK Medical Research Council (C375/A17721 and MR/M000141/1 to E.Y.J.), and Wellcome Trust grant 203141/Z/16/Z supporting the Wellcome Centre for Human Genetics and MICRON imaging facility (http://micronoxford.com, supported by WellcomeStrategic Awards 091911/B/10/Z and 107457/Z/15/Z). We thank K. Channon and G. Douglas for providing the Home Office Project Licences under which part of the animal studies were performed, A. Jefferson for help with confocal imaging, for technical advice and access to equipment, V. Jain for providing the NRP1 plasmid and L. Payne for help with qPCR.
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Contributions
V.M. performed or was involved in most of the experiments and analyses. K.-L.P. performed en face staining and imaging of all aortas, staining and imaging for in vitro alignment, most qPCR experiments and quantification of the data. D.R. designed and validated the ring-locked PLXND1 mutant. K.N. performed activation of signalling mediators in response to shear stress and initial magnetic force application experiments. A.K. performed semaphorin challenge experiments and analysed the data. D.L. performed the calcium-imaging experiments. Y.K. provided structural analysis of the PLXND1 ectodomain. D.K. and M.A. carried out the negative-stain electron-microscopy analysis. J.H. performed the initial PLXND1 siRNA experiments. Y.F. provided the design of the cone-and-plate system. A.d.R.H. led and supervised the calcium-imaging experiments. J.S.R. cosupervised and interpreted data, conceived and developed the idea of the binary conformations of PLXND1 and performed cloning of the PLXND1 wild-type and mutant constructs into adenovirus. E.Y.J. led and supervised the structural-biology-based components of the study. E.T. initiated the project, generated research funds and ideas, directed and coordinated the project. V.M., J.S.R., E.Y.J. and E.T. designed experiments, interpreted data and wrote the manuscript, with inputs from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Knockout or knockdown of PLXND1 and other genes in ECs.
a–f, ECs were either isolated from Plxnd1fl/fl and Plxnd1iECKO mice, or treated with siRNAs to knockdown PLXND1, NRP1, PIEZO1 and Gαq/11. Knockdowns and knockouts were confirmed by western blotting, using GAPDH as a loading control. g, PLXND1 was knocked down in mouse ECs using a pool of siRNAs, followed by infection with an adenovirus expressing either β-galactosidase (LacZ), or wild-type or mutant PLXND1. Protein levels were normalized to GAPDH. KD, mean knockdown efficiency based on n = 3; KO, mean knockout efficiency based on n = 3.
Extended Data Fig. 2 PLXND1 mediates the EC response to fluid shear stress.
a, BAECs were transfected with scrambled or PLXND1 siRNA and exposed to laminar fluid shear stress (12 dynes cm−2) using a parallel plate system for the indicated time periods. Phosphorylation of Akt (n = 6), ERK1/2 (n = 5) and eNOS (n = 8) was determined by western blotting and quantified using Image Studio Lite v.5.2. Data are mean ± s.e.m. P values were obtained by two-tailed Student’s t-tests using GraphPad Prism.*P < 0.05 relative to the static condition; #P < 0.05 relative to the shear time point of the respective scrambled siRNA. b, BAECs were transfected with scrambled or PLXND1 siRNA and exposed to atheroprotective shear stress for 24 h. Cells were fixed and stained with phalloidin, DAPI and anti-β-catenin antibodies to visualize actin stress fibres, nuclei and cell junctions, respectively. Quantification of alignment was performed using ImageJ; n > 50 cells across 4 biological replicates (exact sample numbers are provided in the Source Data). Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism. ****P < 0.0001.
Extended Data Fig. 3 Mechanotransduction by PLXND1 is independent of its ligand-binding functions.
a, BAECs were treated with SEMA3E-blocking antibody or control antibody (1 μg ml−1) and exposed to fluid shear stress for the indicated times. Phosphorylation of eNOS, Akt and ERK1/2 was determined by western blotting and quantified using Image Studio Lite v.5.2. n = 3 biological repeats. Data are mean ± s.e.m. b, BAECs were treated with SEMA3E-blocking antibody or control antibody for 1 h before exposure to SEMA3E for 30 min at the indicated concentrations. Cells were fixed and probed with anti-vinculin antibody, then stained with phalloidin and DAPI to visualize focal adhesions, actin stress fibres and nuclei, respectively. EC collapse was quantified by measuring the cell area using ImageJ. Data are mean ± s.e.m. Significance was determined by ANOVA with a Tukey post hoc test using GraphPad Prism. ****P < 0.0001. n = 59–82 cells across 3 independent experiments (exact sample numbers are provided in the Source Data). Scale bar, 50 μm.
Extended Data Fig. 4 Lipid-profile analysis and expression of inflammatory markers in the aortic arch.
a, Body weights and lipid-profile analysis of Plxnd1fl/flApoe−/− and Plxnd1iECKOApoe−/− mice after 10 weeks of high-fat diet feeding (analysed at 16–17 weeks of age); n = 8. Data are mean ± s.e.m. b, Representative en face preparations of aortic arches immunostained for VCAM-1 (n = 3) and MCP-1 (n = 5) from Plxnd1fl/flApoe−/− and Plxnd1iECKOApoe−/− mice with quantification of fluorescence intensity in fold change; 3–5 images were taken from the inner curvature of aortic arch of each mouse. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism. *P < 0.05.
Extended Data Fig. 5 Atherosclerosis in the descending aorta.
a, Representative en face preparations of the whole aorta showing atherosclerosis in Plxnd1fl/flApoe−/− and Plxnd1iECKOApoe−/− mice after 20 weeks of high-fat diet feeding, visualized by oil-red-O staining. b, Quantification of lesion area in the thoracic aortas, abdominal aortas and whole descending aortas (thoracic aorta and abdominal aorta) of n = 9 Plxnd1fl/flApoe−/− and n = 8 Plxnd1iECKOApoe−/− mice. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism. *P < 0.05, **P < 0.01, ****P < 0.0001.
Extended Data Fig. 6 Mechanical force on PLXND1 results in integrin activation, whereas ligand stimulation causes ECs to collapse.
a, BAECs were incubated with anti-PLXND1-coated beads and subjected to force (10 pN) for 5 min. ECs were fixed and stained with HUTS4 antibody to mark ligated β1 integrin. Mean fluorescence intensity was quantified using ImageJ software. Values were normalized to the no force condition. Locations of the beads are highlighted in yellow circles. n = 50 cells per condition from 3 independent experiments. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism. ****P < 0.0001. Scale bar, 10 μm. b, BAECs were incubated with SEMA3E or vehicle, fixed and stained with anti-vinculin antibody to mark focal adhesions. Focal adhesion number was quantified using ImageJ software. Values were normalized to the vehicle condition. n = 30 cells per condition from 3 independent experiments. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism. ****P < 0.0001. Scale bar, 10 μm.
Extended Data Fig. 7 PLXND1 colocalizes and associates with members of the junctional mechanosensory complex, and its levels are not regulated by flow, in contrast to SEMA3E.
a, The descending thoracic aorta or the inner curvature of aortic arches were isolated and prepared en face from wild-type mice and stained for PLXND1, PECAM-1 and DAPI. Quantification of PLXND1 levels was performed by fluorescence intensity measurement using ImageJ; 4–6 images were taken of tissue collected from n = 4 mice. Data are mean ± s.e.m. Scale bar, 20 μm. b, The descending thoracic aorta was isolated and prepared en face from Plxnd1iECKO mice and stained for PLXND1 expression to assess the specificity of the PLXND1 immunostain. n = 3 mice all showed similar results. c, The descending thoracic aorta or the inner curvature of aortic arches were isolated and prepared en face from wild-type mice and stained for SEMA3E and PECAM-1 expression and with DAPI. Quantification of SEMA3E levels was performed by fluorescence intensity measurement using ImageJ; 4–6 images were taken using tissue collected from n = 3 mice. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-test using GraphPad Prism. *P < 0.05. Scale bar, 20 μm. d, Mouse ECs were exposed to shear stress for the indicated times or left as static controls before immunoprecipitating PLXND1 and analysing its association with the junctional mechanosensory complex (PECAM, VE-cadherin and VEGFR2) as well as PI3K/p85. n = 3 independent experiments.
Extended Data Fig. 8 Relationship between PLXND1 and other established mechanosensors.
a, Pecam1+/+ and Pecam1−/− ECs were incubated with anti-PLXND1-coated beads and subjected to force application for 5 min before the analysis of the phosphorylation of vinculin. n = 3 independent experiments. *P < 0.05. b, c, Mouse ECs were treated with siRNAs against Piezo1 and Gαq/11, incubated with anti-PLXND1-coated beads and subjected to force application for 5 min before the analysis of the phosphorylation of vinculin. n = 3. *P < 0.05. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism.
Extended Data Fig. 9 Force application on other members of the PLXND1 mechanocomplex does not elicit a mechanotransduction response.
a, b, Mouse ECs were incubated with anti-VEGFR2 (a) or anti-NRP1 (b) antibody-coated beads and subjected to force (10 pN) for the indicated time periods. Phosphorylation of Akt and ERK1/2 was determined by western blotting and quantified using Image Studio Lite v.5.2. n = 3 biological repeats. Data are mean ± s.e.m.
Extended Data Fig. 10 Validation of the PLXND1 mutant.
a, Negative-stain two-dimensional class averages of wild-type PLXND1 were obtained by classifying 1,305 particles into 10 classes. Scale bar, 10 nm. b, Negative-stain two-dimensional class averages of mutant PLXND1 were obtained by classifying 1,357 particles into 10 classes. c, The double mutant of PLXND1 was labelled with a thiol-reactive fluorescent dye, Alexa Fluor 488 C5 maleimide. The degree of labelling shows that the vast majority of PLXND1 mutant molecules form the disulfide-linked bond and thus the ring of the majority of PLXND1-mutant molecules appears to be locked by the covalent bond. The degree of labelling for the hen egg ovalbumin, which we used as a positive control, is close to the number of free cysteines in ovalbumin. n = 3 independent experiments. Data are mean ± s.e.m. P values were calculated by two-tailed Student’s t-tests using GraphPad Prism. ***P < 0.001.
Extended Data Fig. 11 An open conformation of PLXND1 is required for force-dependent signalling.
Mouse lung ECs in which endogenous PLXND1 was knocked down were infected with adenoviruses expressing β-galactosidase (Ad.LacZ), wild-type or mutant PLXND1 and incubated with anti-PLXND1 paramagnetic beads, followed by force application for 5 min before lysing and assaying vinculin phosphorylation by western blotting. n = 3 biological repeats. Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests using GraphPad Prism. *P < 0.05.
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.Uncropped western blot scans with size marker indications.
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Mehta, V., Pang, KL., Rozbesky, D. et al. The guidance receptor plexin D1 is a mechanosensor in endothelial cells. Nature 578, 290–295 (2020). https://doi.org/10.1038/s41586-020-1979-4
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DOI: https://doi.org/10.1038/s41586-020-1979-4
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