Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation

Abstract

Mutations in the transcription factor FOXC2 are predominately associated with lymphedema. Herein, we demonstrate a key role for related factor FOXC1, in addition to FOXC2, in regulating cytoskeletal activity in lymphatic valves. FOXC1 is induced by laminar, but not oscillatory, shear and inducible, endothelial-specific deletion impaired postnatal lymphatic valve maturation in mice. However, deletion of Foxc2 induced valve degeneration, which is exacerbated in Foxc1; Foxc2 mutants. FOXC1 knockdown (KD) in human lymphatic endothelial cells increased focal adhesions and actin stress fibers whereas FOXC2-KD increased focal adherens and disrupted cell junctions, mediated by increased ROCK activation. ROCK inhibition rescued cytoskeletal or junctional integrity changes induced by inactivation of FOXC1 and FOXC2 invitro and vivo respectively, but only ameliorated valve degeneration in Foxc2 mutants. These results identify both FOXC1 and FOXC2 as mediators of mechanotransduction in the postnatal lymphatic vasculature and posit cytoskeletal signaling as a therapeutic target in lymphatic pathologies.

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity

“…Although in vivo validation remains to be done, our findings support a concept that continuous basal activation of EphrinB2/EphB4 signalling provides a homeostatic control of Rac1/Rho-mediated cytoskeletal contractility to regulate LEC junction integrity. Interestingly, loss of (valve-specific) FOXC2 and FOXC1 in LECs was recently shown to result in increased actin stress fibre formation and Rho activity, suggesting similar mechanisms of junctional regulation in valve LECs ( Norden et al, 2020 ). It may therefore not be surprising that ablation of the adhesion molecule CLDN5 was not sufficient to induce a breakdown of LEC junctions in the absence of activation of the cytoskeleton.…”

Molecular Mechanisms Controlling Lymphatic Endothelial Junction Integrity

“…A recent study further demonstrates a complementary role of FOXC1 in addition to FOXC2 as key mediators of mechanotransduction in the regulation of LEC junctional integrity (Norden et al, 2020 ). Unlike FOXC2, FOXC1 is not increased in LECs by OSS, but by laminar shear stress (LSS), and FOXC1 is highly enriched in LECs located at the leading free-edge of the intraluminal side of valve leaflets that are exposed to LSS in mesenteric lymphatic valves of the adult mice (Norden et al, 2020 ). Inducible endothelial cell (EC)-specific Foxc1 deletion in mice impairs postnatal lymphatic valve maturation, whereas EC-deletion of Foxc2 induces valve degeneration, which is exacerbated in EC-specific compound Foxc1 and Foxc2 mutant mice.…”

“…Treatment of cells with the ROCK inhibitor Y-27632 was able to rescue this impaired phenotype and restore continuous junctions in HDLECs. It is postulated that transcriptional regulation of a molecular signaling complex, consisting of the planar cell polarity signaling component PRICKLE1 and the RhoA GTPase activating proteins (GAPs) Arhgap21 and Arhgap23, by FOXC1 and FOXC2 negatively regulates downstream Rho/ROCK signaling as previously described (Norden et al, 2020 ). (C) siRNA-mediated knockdown of the ligand EphrinB2, subsequently resulting in impaired signaling through its receptor EphB4, was shown to reduce Rac1 GTPase activation in HDLECs.…”

“…In the blood endothelium, VEGF-A/VEGFR-2-mediated activation of the small GTPase RhoA/Rho-associated protein kinase (ROCK) signaling leads to cytoskeletal rearrangement of cortical actin into perpendicular stress fibers binding to VE-cadherin, thereby regulating cell junctions and vascular permeability (Dejana et al, 2009b ; Dorland and Huveneers, 2017 ; Szymborska and Gerhardt, 2018 ). As described below, accumulating evidence suggest that the RhoA/ROCK pathway controls LEC junction integrity (Zhang et al, 2018 ; Frye et al, 2020 ; Norden et al, 2020 ).…”

Lymphatic Valves and Lymph Flow in Cancer-Related Lymphedema

“…Even genes previously identified to cause congenital lymphedema that were not initially reported to exhibit valve defects in the knockout models have recently been demonstrated to harbor valve defects (e.g., RASA1 [ 75 , 76 ]). The presence of valves in animal models should not be assumed to imply the absence of valve defects because mutations can cause a reduction, not a complete loss, in the number of valves and/or reduce the valve leaflet length (e.g., FOXC2 [ 77 ]). Given that valve defects are such a common theme in congenital lymphedema, it is logical to suggest that defective valves may also play a significant role in the pathophysiology of CRL.…”