A non-canonical Notch complex regulates adherens junctions and vascular barrier function

Abstract

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis1. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function2, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial–venous differentiation3, angiogenesis4 and remodelling5. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

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Figure 1: NOTCH1 regulates shear stress-induced vascular barrier function.
Figure 2: The NOTCH1 transmembrane domain mediates barrier function through interaction with VE-cadherin.
Figure 3: NOTCH1 assembles a mechanosensory junctional complex involving LAR, TRIO, and RAC.

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health (EB00262, EB08396, UH3EB017103 and HL115553) and the National Science Foundation Center for Engineering MechanoBiology (CMMI15-48571). W.J.P. acknowledges support from a Ruth L. Kirchstein National Research Service Award (F32 HL129733) and from the NIH through the Organ Design and Engineering Training program (T32 EB16652), and M.L.K. acknowledges support from the Hartwell Foundation and the NIH through the Translational Research in Regenerative Medicine Training program (T32 EB005583). We thank P. A. Murphy and R. Wang for discussions of preliminary data and M. Schwartz for materials and discussions.

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Authors

Contributions

W.J.P., M.L.K., and C.S.C. conceived the study and designed experiments. W.J.P. and M.L.K. performed all experiments and data analysis with assistance from J.E. (qPCR) and J.Y. (Notch1-knockout mice). Y.W., H.V. and K.K.H. provided Notch1-knockout mice. W.J.P., M.L.K. and C.S.C. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Christopher S. Chen.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks H. Gerhardt, I. Geudens, E. Tzima, A. Yap, and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Known permeability-modulating agents regulate barrier function in hEMVs.

ad, Diffusive permeability measured in hEMVs in static or flow conditions treated with 500 nM S1P for 1 h (a), 10 μM SU415 (VEGFR2 inhibitor) overnight (b), 1 U ml−1 thrombin for 15 min (c), or 40 ng ml−1 VEGF for 1 h (d). e, Western blots of cleaved NOTCH1 ICD (using N1 V1754 antibody) from lysates of endothelial cells treated with vehicle or 40 ng ml−1 VEGF for 1 h. All plots show mean ± s.e.m., n ≥ 3 hEMVs, **P < 0.01. Exact P and n values are shown in Source Data; western blot is representative of two independent experiments. Source data

Extended Data Figure 2 Flow activates expression of mechanotransduction-associated gene networks.

Relative gene expression measured by qPCR. –ΔΔCt of GOI under flow normalized to static control is plotted as a heat map (n = 3 flow or static qPCR analyses from distinct hEMV sets; each column is representative of an independent experiment; *P < 0.05, **P < 0.01). Exact P and n values are shown in Source Data. Source data

Extended Data Figure 3 Non-transcriptional NOTCH1 signalling regulates vascular barrier function.

a, Expression of NOTCH1-target genes HES1 and HEY1 and the genes for the NOTCH1 ligand (DLL4) and VE-cadherin (CDH5) measured by qPCR in endothelial cells treated with DAPT or DMSO-load control on DLL4-coated and control tissue culture plastic substrates. b, Fluorescent micrograph of DLL4-coated device before cell seeding (green, Alexa Fluor 488–collagen I; red, DLL4 immunofluorescence). c, Fluorescent micrograph of endothelial cells in hEMVs coated with DLL4 before cell seeding. d, Micrographs of GFP-infection control cells under flow. e, Gene expression of HES1, HEY1, DLL4, NOTCH1, and CDH5 measured by qPCR in endothelial cells expressing dominant-negative MAML (dnMAML) or infection control (GFP). f, Western blot validation of NOTCH1 CRISPR lines: Scramble, NOTCH1KO, TMD-ICD KO, and ICD KO. g, Fluorescent micrographs of CRISPR–Cas9 scramble control cells cultured in flow conditions. h, Fluorescent micrographs of scramble control and NOTCH1KO hEMVs cultured in static conditions, immunostained for VE-cadherin and labelled with phalloidin. i, Quantification of junctional area measured from micrographs of cells immunostained for VE-cadherin as described in h. j, Gene expression measured by qPCR in NOTCH1KO and scramble control cells. k, Quantification of cell number in field of view at 10× magnification of scramble or NOTCH1KO hEMVs cultured in static and flow conditions. l, Micrographs of nuclei as visualized by DAPI in scramble or NOTCH1KO hEMVs. For all plots, mean ± s.e.m., n ≥ 3 hEMVs, **P < 0.01. Exact P and n values are shown in Source Data; images are representative of at least three independent experiments. Source data

Extended Data Figure 4 NOTCH1 is activated in response to shear stress by endocytosis of DLL4.

a, ICD cleavage as measured by western blot with an antibody specific to cleaved ICD (N1 V1754) in DLL4KO and scramble control endothelial cells cultured under flow. b, Fluorescent micrographs of DLL4KO and scramble control hEMVs cultured under flow conditions, immunostained for VE-cadherin and labelled with phalloidin. c, Quantification of junctional area measured from micrographs of cells immunostained for VE-cadherin. d, Fluorescence micrograph of endothelial cells expressing DLL4–HA in static plus DMSO, flow plus DMSO, and flow plus Dynasore conditions, stained for haemagglutinin and DAPI. e, Quantification of internalized DLL4–HA in endothelial cells under static plus DMSO, flow plus DMSO, and flow plus Dynasore conditions. Cells with more than one Alexa Fluor 488-positive punctum counted as positive for internalized DLL4–HA. f, Immunofluorescence of an endothelial cell expressing DLL4–HA cultured under flow and stained for haemagglutinin and NOTCH1 ECD and stained with DAPI. g, Diffusive permeability of 70-kDa dextran in cells treated with Dynasore hydrate or DMSO-load control and exposed to flow overnight. All plots show mean ± s.e.m., n ≥ 3 hEMVs, **P < 0.01. Exact P and n values are given in Source Data; images are representative of at least two independent experiments. Source data

Extended Data Figure 5 DAPT and NOTCH1 depletion modulate vascular permeability in vivo.

a, Diffusive permeability of mouse dermal vasculature as a function of vessel diameter after 1 h of intravenously injected DMSO or DAPT (n = 15 vessels across 3 mice per condition). b, High magnification whole-mount micrographs of Evans blue in the mouse dermal vasculature. Fluorescent images are representative of three independent experiments. Source data

Extended Data Figure 6 NOTCH1 regulates junctional stability through association with VE-cadherin.

a, Time-lapse images of cells expressing VE-cadherin–mApple in the presence of DAPT or DMSO load control demonstrate that adherens junctions disassemble after 30 min of exposure to DAPT, leading to macroscopic intercellular gaps (red arrows). b, Fluorescent micrographs of NOTCH1KO cells expressing TMD–ICD–mApple or TMD–ICD(V1754G)–mApple immunostained for cleaved NOTCH1 (ICD V1754) and stained with DAPI. c, Fluorescent micrographs of TMD–mApple expressed in CHD5KO or scramble control endothelial cells and immunostained for VE-cadherin. d, Western blot for NOTCH1 ICD and VE-cadherin in NOTCH1KO and CHD5KO endothelial cells. e, Western blot validation of NOTCH1 rescue constructs: mApple, TMD–mApple, ICD–TMD–mApple, and ICD–TMD(V1754G)–mApple. f, Immunoprecipitation of VE-cadherin from hMVEC-D cells treated with DMSO or DAPT. Co-immunoprecipitation of mechanosensory complex components was assessed by immunoblotting for NOTCH1 ICD, TRIO, and LAR. g. Western blot of VE-cadherin immunoprecipitations from NOTCH1KO cells expressing NOTCH1-TMD truncation constructs (6, 8 and 12 amino acids from the N terminus) fused to mApple. h, Western blot of VE-cadherin immunoprecipitations from NOTCH1KO cells expressing NOTCH1 TMD constructs with single and double point mutations within the TMD and fused to mApple. Images are representative of at least three independent experiments.

Extended Data Figure 7 DLL4 and the NOTCH1 mechanosensory complex are critical for increased RAC1 activity in response to shear stress.

a, Active RAC1 was isolated from hMVEC-D cell lysates treated with DMSO and DAPT (20 μM) using a pull-down assay with GST–PBD. b, Quantification of band intensity from a demonstrates a decrease (~30%) in RAC1 activity with DAPT treatment. c, Active RAC1 was isolated from hMVEC-D cell lysates from static or shear flow conditions using GST–PBD pull-down. d, Active RAC1 was isolated using GST–PBD pull-down from DLL4KO cell lysates under flow conditions. e, Active RAC1 was isolated using GST–PBD pull-down from NOTCH1KO, PTPRFKO and TRIOKO cell lysates under flow conditions. Mean ± s.e.m., n = 3 independent lysates, **P < 0.01. Exact P and n values are shown in Source Data; all images are representative of at least three independent experiments. Source data

Extended Data Figure 8 NOTCH1 regulates VE-cadherin-interacting proteins to form the NOTCH1 mechanosensory complex.

a, Immunoprecipitation of VE-cadherin from NOTCH1KO and scramble control cells. Co-immunoprecipitation of candidate NOTCH1-dependent VE-cadherin effectors was assessed by immunoblotting for VE-PTP, VEGFR2 and LAR (85 kDa P subunit). b, Immunoprecipitation of TRIO from scramble control, NOTCH1KO, and PTPRFKO cells. Immunoblotting for VE-cadherin was used to assess impaired TRIO–VE-cadherin co-immunoprecipitation on depletion of NOTCH1 or LAR. c, Western blots of VE-cadherin immunoprecipitates of lysates from lungs of Cdh5-cre+:Notch1fl/fl and control Cdh5-cre:Notch1fl/fl mice. d, Western blots of Trio immunoprecipitates from lysates of lungs from Cdh5-cre+:Notch1fl/fl and control Cdh5-cre:Notch1fl/fl mice. e, Western blot of proximal interacting proteins extracted with streptavidin from hMVEC-D cells expressing BIRA–HA (BioID) or VE-cadherin–BIRA–HA (VE-BioID) that were treated with DLL4, DAPT or DMSO. f, Western blot of proximal interacting proteins extracted with streptavidin in hMVEC-D cells expressing VE-cadherin–BIRA–HA (VE-BioID) that were treated with DLL4, DAPT or DMSO. Images are representative of at least two independent experiments.

Extended Data Figure 9 The NOTCH1 mechanosensory complex stabilizes cell–cell junctions through activation of RAC1.

Flow induces endocytosis of DLL4, triggering the activation and cleavage of NOTCH1 ICD and ECD, which allows the NOTCH1 TMD to link the adaptor protein LAR with VE-cadherin and recruit TRIO to adherens junctions. The resulting complex activates RAC1, elaborates cortical actin, and stabilizes cell–cell junctions to establish barrier function.

Extended Data Figure 10 Supplementary methods.

a, Schematic and summary of methods for quantifying vascular permeability in vivo with intravital microscopy. b, Intensity as a function of distance along lines connecting the nucleus centroids of neighbouring cells. Blue circles are local maxima used to count the number of stress fibres per unit length, and the shaded region is the area under the peak corresponding to cortical actin, and is normalized against the total area under the curve for quantification. The graph is representative of three independent experiments.

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This file contains Supplementary Tables 1-2 and the uncropped western blot scans with size marker indications. (PDF 2940 kb)

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Polacheck, W., Kutys, M., Yang, J. et al. A non-canonical Notch complex regulates adherens junctions and vascular barrier function. Nature 552, 258–262 (2017). https://doi.org/10.1038/nature24998

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