Mechanoreceptor plexin D1 regulates lymphatic valve morphogenesis and lymphedema pathogenesis
Kar-Lai Pang, Vedanta Mehta, Claire Aitken, Sara E. Dobbins, Jing Yu, Gabriele Bonetti, Adam N. Keen, Feiran Zhang, Amélie Sabine, Tatiana V. Petrova, Paul R. Riley, E. Yvonne Jones, Sandro Michelini, Matteo Bertelli, John S. Reader, Pia Ostergaard, Ellie Tzima
Kar-Lai Pang, Vedanta Mehta, Claire Aitken, Sara E. Dobbins, Jing Yu, Gabriele Bonetti, Adam N. Keen, Feiran Zhang, Amélie Sabine, Tatiana V. Petrova, Paul R. Riley, E. Yvonne Jones, Sandro Michelini, Matteo Bertelli, John S. Reader, Pia Ostergaard, Ellie Tzima
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Research Article Cell biology Vascular biology

Mechanoreceptor plexin D1 regulates lymphatic valve morphogenesis and lymphedema pathogenesis

Abstract

Lymphatic valves are essential for maintaining tissue fluid homeostasis, and their dysfunction leads to lymphedema, a morbid and disfiguring disease without a cure. Mechanical forces due to lymph flow are required for proper lymphatic valve development, yet it remains unclear how lymphatic endothelial cells (LECs) sense and decode mechanical signals. In this study, we identify the cell guidance semaphorin receptor plexin D1 (PLXND1) as a lymphatic mechanosensor required for lymphatic valve morphogenesis. Conditional genetic ablation of Plxnd1 in LECs caused major defects in lymphatic valve development in 2 different lymphatic vascular beds. Mechanistically, PLXND1 acted as a mechanosensor within a lymphatic mechanocomplex, initiating distinct mechanical signals and activating the lymphatic valve transcriptional program through an unconventional pathway. Screening of patients with primary lymphedema identified PLXND1 missense variants, and functional analysis established 2 pathogenic variants that selectively disrupt the ligand versus mechanosensing functions of this receptor. Variants associated with lymphedema in members of the mechanocomplex disrupted its formation, underscoring the central role of this complex in lymphatic valve biology. Our work uncovers a mechanosensing mechanism guiding lymphatic valve development, and has profound implications for the understanding and treatment of primary lymphedema in humans.

Authors

Kar-Lai Pang, Vedanta Mehta, Claire Aitken, Sara E. Dobbins, Jing Yu, Gabriele Bonetti, Adam N. Keen, Feiran Zhang, Amélie Sabine, Tatiana V. Petrova, Paul R. Riley, E. Yvonne Jones, Sandro Michelini, Matteo Bertelli, John S. Reader, Pia Ostergaard, Ellie Tzima

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Figure 5

PLXND1 variants associated with lymphedema selectively disrupt ligand versus mechanical activation of the receptor.

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PLXND1 variants associated with lymphedema selectively disrupt ligand v...
(A) Lollipop plot of PLXND1 variants showing a 2D representation of their location in the PLXND1 protein. The red variant (S178N) resides within the SEMA binding domain, the green variant (Q676E) is located distal from this domain, and the blue variants correspond to mutations that have not yet been functionally characterized. PSI, plexin-semaphorin-integrin homology domains; IPT/TIG, immunoglobulin plexin transcription/transcription factor immunoglobulin domains; TM, transmembrane domain. (B) Schematic showing ligand activation of PLXND1 via SEMA3E and the downstream signaling investigated. (C) SEMA3E-induced cell collapse assays. HLECs in which endogenous PLXND1 was knocked down were transduced with adenoviruses expressing WT or mutant Q676E or S178N PLXND1 and were subsequently treated with SEMA3E and immunostained with anti-vinculin antibody (cyan) and phalloidin (magenta). Representative images from 3 biological replicates are shown. Scale bar: 20 μm. (D) HLECs in which endogenous PLXND1 was knocked down were transduced with adenoviruses expressing WT or mutant PLXND1 and treated with SEMA3E before assaying phosphorylation of FAK (pY397) and cofilin (pS3) (n = 3, data are the ± SEM; 2-way ANOVA with Tukey’s multiple-comparison test, ****P < 0.0001). (E) Schematic showing mechanical activation of PLXND1 via tensional force application using magnetic beads and the downstream signaling investigated. (F) HLECs in which endogenous PLXND1 was knocked down were transduced with adenoviruses expressing WT or mutant PLXND1 and incubated with anti-PLXND1 magnetic beads followed by force application. Phosphorylation of CDK5 (pY15) was determined (n = 3, data are the mean ± SEM; 2-way ANOVA with Tukey’s multiple-comparison test, *P < 0.05).