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 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 2

Lymphatic valve development is blocked in Clec2–/– mesenteric lymphatics.

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Lymphatic valve development is blocked in Clec2–/– mesenteric lymphatics...
(A–D) Whole-mount staining for PROX1 in P1 neonatal mesentery was used to identify lymphatic valves. White arrows indicate PROX1HI lymphatic valves. Scale bars: 200 μm. Representative images shown from 6 mice per genotype. (E and F) Analysis of venous valves in the femoral vein of P6 pups by visualization of PROX1-GFP, which is expressed in venous valve BECs. White arrows indicate venous valves. Representative images shown from 4 mice per genotype. (G) Quantitation of lymphatic valve number in neonatal Clec2–/– and Clec2+/+ littermates. n = 6 mice per genotype. (H) Quantitation of lymphatic valve number in neonatal platelet-specific conditional Clec2fl/–; Pf4-Cre and Clec2 fl/+; Pf4-Cre littermates. n = 3 mice per genotype. All values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, calculated by Student’s t test. (K–T) Analysis of SMC coverage of Clec2–/– and Clec2+/+ littermates. (I–N) Staining of mesenteric vessels for smooth muscle actin (red) and lymphatic ECs (PROX1-GFP, green) in P4 neonates. V, vein; A, artery; L, lymphatic. White dotted line in K–L outlines lymphatic vessels. White arrows indicate lymphatic valves. Scale bars: 200 μm. Representative images shown from 3 mice per genotype. (O–R) High-magnification confocal microscopy of P4 lymphatic valve regions (smooth muscle actin, red) and lymphatic ECs (PROX1-GFP, green). V, valve. Scale bars: 100 μm. (S and T) Histology of E18.5 thoracic duct (TD) for smooth muscle actin (red) and lymphatic ECs (PROX1-GFP, green). Scale bars: 100 μm. Representative images shown from 2 mice per genotype.