Repression of MAPK/Erk signaling by Efnb2-Ephb4-Rasa1 is required for lymphatic valve formation

The lymphatic vascular system plays important roles in various physiological and pathological processes, and lack of lymphatic or lymphovenous valves always causes lymph or blood reflux, and can lead to lymphedema. However, the molecular mechanism underlying the valve formation is poorly understood. Here we report that the MAPK/Erk signaling needs to be repressed during the valve-forming lymphatic endothelial cells (LECs) fate determination, which differs from its positive role in the LECs specification. Up-regulation of MAPK/Erk signaling in ephb4b, efnb2a;efnb2b and rasa1a;rasa1b mutants leads to lymphatic valve defects, whereas simultaneous loss of Erk1 and Erk2 causes valve hyperplasia. Moreover, valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated in the presence of MEK inhibitors, indicating a new function of Efnb2-Ephb4-Rasa1 cassette in lymphatic valve progenitor cells specification by repressing MAPK/Erk activity. Therefore, our findings provide a mechanistic understanding of the lymphatic valve formation and potential drug targets for related lymphatic diseases.

Numerical Study of Lymph Mechanics

Methods taken from engineering and computer science were applied to the lymphatic system. Starting with a 3D analysis of a single subject-specific lymphatic valve. A mechanism was presented to explain previous experimental results showing the effect of trans-mural pressure on the pressure required to close lymphatic valves. The impor-tance of wall motion in future FSI studies of lymphatic valve dynamics were identified. Previous approaches to lumped modelling of the lymphatic system were considered and modifications were proposed. A less-idealised valve model, incorporating trans-mural dependent bias, was proposed as well as a method of allowing self-organised contrac-tion through a stretch-dependent frequency of contraction. A network of the superficial lymphatics of the upper-limb was reconstructed from an anatomical sketch. The net-work was used in conjunction with the lumped model to produce a 421 vessel lymphatic model consisting of 17,706 lymphangions. Several issues which impede large network scale modelling of the lymphatic system are identified. A simplified patient-specific biphasic model of lymphoedema was proposed and used to develop a novel shape-based metric for lymphoedema. A statistically significant relationship between the metric and the presence of lymphoedema was found.

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

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.