Lymph flow regulates collecting lymphatic vessel maturation in vivo
Daniel T. Sweet, Juan M. Jiménez, Jeremy Chang, Paul R. Hess, Patricia Mericko-Ishizuka, Jianxin Fu, Lijun Xia, Peter F. Davies, Mark L. Kahn
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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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 5

Lymphatic shear stress induces the expression of most genes required for lymphatic valve development.

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Lymphatic shear stress induces the expression of most genes required for...
(A) The reversing flow regimen shown was used to expose human LECs to fluid shear forces known to exist in lymphatic collecting vessels in vivo for 48 hours. LECs were exposed to a maximum of 3.25 dynes/cm2, a minimum of –1.25 dynes/cm2, and an average 0.67 dynes/cm2 of shear stress. (B and C) Changes in the expression of genes associated with lymphatic valve development (B) and genes known to be induced by laminar shear in blood ECs (C) were measured as fold-change compared with static control and normalized to GAPDH following LEC exposure to the lymphatic flow regimen shown in (A). n = 4 experiments. All values expressed as fold-change means ± SEM. *P < 0.05, calculated by Student’s t test. (D) Western blot showing upregulation of protein expression in response to 48-hour lymphatic shear. Blots were probed for GATA2, FOXC2, and CX37, with GAPDH as a loading control. Arrows indicate slight molecular weight shift of FOXC2 after flow. n = 3 experiments.