Lymph flow regulates collecting lymphatic vessel maturation in vivo
Daniel T. Sweet, … , Peter F. Davies, Mark L. Kahn
Daniel T. Sweet, … , Peter F. Davies, Mark L. Kahn
Published August 3, 2015; First published July 27, 2015
Citation Information: J Clin Invest. 2015;125(8):2995-3007. https://doi.org/10.1172/JCI79386.
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Research Article Angiogenesis Cardiology Development Oncology Vascular biology

Lymph flow regulates collecting lymphatic vessel maturation in vivo

Abstract

Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin–like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development.

Authors

Daniel T. Sweet, Juan M. Jiménez, Jeremy Chang, Paul R. Hess, Patricia Mericko-Ishizuka, Jianxin Fu, Lijun Xia, Peter F. Davies, Mark L. Kahn

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Figure 1

Lack of detectable lymph flow in developing mesenteric lymphatics of Clec2–/– mice.

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Lack of detectable lymph flow in developing mesenteric lymphatics of Cle...
(A–F) Clec2–/– mice exhibit edema in the intestine wall after E15.5. H&E staining of cross sections of embryonic intestine is shown at E15.5, E16.5, and E18.5. Scale bars: 100 μm. (G and H) LEC elongation in P1 mesenteric lymphatics was measured by staining for PROX1 (green) and VE-cadherin (red). Yellow lines indicate representative measurements of cell length and width. Scale bars: 50 μm. (I) Quantitation of intestine wall thickness. n = 2 mice per timepoint for each genotype and 2 sections per mouse with at least 3 wall thickness measurements per section. (J) Quantitation of LEC elongation using length/width ratio. n = 4 animals at E15.5 and 5 animals at P1 per genotype. Over 100 LECs were measured per mouse. All values are means ± SEM. *P < 0.05, ***P < 0.001. P value calculated by Student’s t test. (K–N) Oral lymphangiogram 2 hours after neonatal ingestion of Bodipy-FL C16. White arrows indicate lymphatic vessels in the mesentery. Images are representative of 5 mice per genotype. (O and P) In vivo measurement of lymphatic flow by bead tracking in surviving 4-week-old in Clec2fl/fl vs. Clec2fl/fl; Pf4-Cre mice with blood-filled lymphatic phenotype. Yellow circles indicate position of bead in each frame of video over one contraction cycle. (Q) Quantitation of bead displacement (μm) over time relative to starting point in Clec2fl/fl (black asterisks) vs. Clec2fl/fl; Pf4-Cre (white circles). Representative images shown from bead tracking in 3 mice per genotype.