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 2

PLXND1 regulates the lymphatic valve program via an unconventional pathway.

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PLXND1 regulates the lymphatic valve program via an unconventional pathw...
(A) Schematic of the lymphatic shear stress profile. Human LECs (HLECs) transfected with either scrambled or PLXND1 siRNAs were left static or exposed to shear stress for 48 hours, RNA was isolated, and quantitative PCR (qPCR) was performed to quantify the expression of GJA4 (n = 7, data are the mean ± SEM; 2-way ANOVA with Tukey’s multiple-comparison test, ****P < 0.0001). (B) HLECs transfected with scrambled or PLXND1 siRNAs were left static or exposed to shear stress for 48 hours. Cells were fixed and immunostained to quantify the expression of CX37 (300–350 cells were analyzed per condition; n = 5 biological replicates; 2-way ANOVA with Tukey’s multiple-comparison test, *P < 0.05). Scale bar: 50 μm. (C) Immunofluorescence staining of Plxnd1fl/fl and Plxnd1iLECKO E18.5 mesentery for CX37 (cyan) and PROX1 (magenta). A, artery, L, lymphatic vessel. Arrowheads indicate lymphatic valves (n = 4, scale bar: 40 μm). (D) HLECs transfected with either scrambled, PLXND1, or CDK5 siRNAs were left static or exposed to shear stress before immunoprecipitating FOXC2 and analyzing serine phosphorylation (n = 3). Immunoprecipitation with nonspecific IgG was used as a control. WCL, whole-cell lysate. (E) Phosphorylation of CDK5 (pY15) in HLECs transfected with scrambled or PLXND1 siRNAs and exposed to shear stress (n = 4, data are the mean ± SEM; 2-way ANOVA with Tukey’s multiple-comparison test, *P < 0.05, **P < 0.01). Dashed line indicates where the blot was spliced. (F) HLECs transfected with either scrambled or CDK5 siRNAs were left static or exposed to shear stress for 48 hours, and qPCR was performed to quantify the expression of GJA4 (n = 3, data are the mean ± SEM; 2-way ANOVA with Tukey’s multiple-comparison test, *P < 0.05, **P < 0.01).