The guidance receptor plexin D1 is a mechanosensor in endothelial cells

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: PLXND1 mediates the EC response to fluid shear stress and regulates the site-specific distribution of atherosclerosis.
Fig. 2: PLXND1 is a mechanosensor that mediates the EC response to force.
Fig. 3: The PLXND1, NRP1 and VEGFR2 mechanocomplex functions upstream of known mechanosensory hotspots and is sufficient for the response to shear stress.
Fig. 4: PLXND1 flexion is required for mechanotransduction.

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. 14 and Extended Data Figs. 211 are provided with the paper. Gel source data can be found in Supplementary Fig. 1.

References

  1. 1.

    Davies, P. F. Flow-mediated endothelial mechanotransduction. Physiol. Rev. 75, 519–560 (1995).

  2. 2.

    Givens, C. & Tzima, E. Endothelial mechanosignaling: does one sensor fit all? Antioxid. Redox Signal. 25, 373–388 (2016).

  3. 3.

    Sakurai, A. et al. Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras. Mol. Cell. Biol. 30, 3086–3098 (2010).

  4. 4.

    Aghajanian, H. et al. Semaphorin 3d and semaphorin 3e direct endothelial motility through distinct molecular signaling pathways. J. Biol. Chem. 289, 17971–17979 (2014).

  5. 5.

    Jongbloets, B. C. & Pasterkamp, R. J. Semaphorin signalling during development. Development 141, 3292–3297 (2014).

  6. 6.

    Hahn, C. & Schwartz, M. A. Mechanotransduction in vascular physiology and atherogenesis. Nat. Rev. Mol. Cell Biol. 10, 53–62 (2009).

  7. 7.

    Kong, Y. et al. Structural basis for plexin activation and regulation. Neuron 91, 548–560 (2016).

  8. 8.

    SenBanerjee, S. et al. KLF2 is a novel transcriptional regulator of endothelial proinflammatory activation. J. Exp. Med. 199, 1305–1315 (2004).

  9. 9.

    Hamik, A. et al. Kruppel-like factor 4 regulates endothelial inflammation. J. Biol. Chem. 282, 13769–13779 (2007).

  10. 10.

    Wu, C. et al. Mechanosensitive PPAP2B regulates endothelial responses to atherorelevant hemodynamic forces. Circ. Res. 117, e41–e53 (2015).

  11. 11.

    Zhang, S. H., Reddick, R. L., Piedrahita, J. A. & Maeda, N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258, 468–471 (1992).

  12. 12.

    Collins, C. et al. Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin–RhoA pathway. Curr. Biol. 22, 2087–2094 (2012).

  13. 13.

    Tzima, E. et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437, 426–431 (2005).

  14. 14.

    Li, J. et al. Piezo1 integration of vascular architecture with physiological force. Nature 515, 279–282 (2014).

  15. 15.

    Xu, J. et al. GPR68 senses flow and is essential for vascular physiology. Cell 173, 762–775 (2018).

  16. 16.

    Bays, J. L., Campbell, H. K., Heidema, C., Sebbagh, M. & DeMali, K. A. Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat. Cell Biol. 19, 724–731 (2017).

  17. 17.

    Katsumi, A., Orr, A. W., Tzima, E. & Schwartz, M. A. Integrins in mechanotransduction. J. Biol. Chem. 279, 12001–12004 (2004).

  18. 18.

    Jalali, S. et al. Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands. Proc. Natl Acad. Sci. USA 98, 1042–1046 (2001).

  19. 19.

    Liu, Y., Sweet, D. T., Irani-Tehrani, M., Maeda, N. & Tzima, E. Shc coordinates signals from intercellular junctions and integrins to regulate flow-induced inflammation. J. Cell Biol. 182, 185–196 (2008).

  20. 20.

    Albarrán-Juárez, J. et al. Piezo1 and Gq/G11 promote endothelial inflammation depending on flow pattern and integrin activation. J. Exp. Med. 215, 2655–2672 (2018).

  21. 21.

    Wang, S. et al. Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J. Clin. Invest. 126, 4527–4536 (2016).

  22. 22.

    dela Paz, N. G., Melchior, B. & Frangos, J. A. Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells. Am. J. Physiol. Cell Physiol. 312, C428–C437 (2017).

  23. 23.

    dela Paz, N. G. & Frangos, J. A. Rapid flow-induced activation of Gαq/11 is independent of Piezo1 activation. Am. J. Physiol. Cell Physiol. 316, C741–C752 (2019).

  24. 24.

    Gay, C. M., Zygmunt, T. & Torres-Vázquez, J. Diverse functions for the semaphorin receptor PlexinD1 in development and disease. Dev. Biol. 349, 1–19 (2011).

  25. 25.

    Chauvet, S. et al. Gating of Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to attraction during brain development. Neuron 56, 807–822 (2007).

  26. 26.

    Siebold, C. & Jones, E. Y. Structural insights into semaphorins and their receptors. Semin. Cell Dev. Biol. 24, 139–145 (2013).

  27. 27.

    Suzuki, K. et al. Structure of the plexin ectodomain bound by semaphorin-mimicking antibodies. PLoS ONE 11, e0156719 (2016).

  28. 28.

    Osawa, M., Masuda, M., Kusano, K. & Fujiwara, K. Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction: is it a mechanoresponsive molecule? J. Cell Biol. 158, 773–785 (2002).

  29. 29.

    Collins, C. et al. Haemodynamic and extracellular matrix cues regulate the mechanical phenotype and stiffness of aortic endothelial cells. Nat. Commun. 5, 3984 (2014).

  30. 30.

    Boselli, F., Freund, J. B. & Vermot, J. Blood flow mechanics in cardiovascular development. Cell. Mol. Life Sci. 72, 2545–2559 (2015).

  31. 31.

    McCormick, M. E. & Tzima, E. Pulling on my heartstrings: mechanotransduction in cardiac development and function. Curr. Opin. Hematol. 23, 235–242 (2016).

  32. 32.

    Gitler, A. D., Lu, M. M. & Epstein, J. A. PlexinD1 and semaphorin signaling are required in endothelial cells for cardiovascular development. Dev. Cell 7, 107–116 (2004).

  33. 33.

    Zhang, Y. et al. Tie2cre-mediated inactivation of plexinD1 results in congenital heart, vascular and skeletal defects. Dev. Biol. 325, 82–93 (2009).

  34. 34.

    Baeyens, N. et al. Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling. Proc. Natl Acad. Sci. USA 111, 17308–17313 (2014).

  35. 35.

    Sabine, A. et al. Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation. Dev. Cell 22, 430–445 (2012).

  36. 36.

    Horzum, U., Ozdil, B. & Pesen-Okvur, D. Step-by-step quantitative analysis of focal adhesions. MethodsX 1, 56–59 (2014).

  37. 37.

    Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  38. 38.

    Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D 62, 1243–1250 (2006).

  39. 39.

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

Download references

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.

Author information

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.

Correspondence to Ellie Tzima.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Knockout or knockdown of PLXND1 and other genes in ECs.

af, 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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

Supplementary information

Supplementary Figure 1

.Uncropped western blot scans with size marker indications.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mehta, V., Pang, K., 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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.