Adrenomedullin signaling is necessary for murine lymphatic vascular development
Kimberly L. Fritz-Six, … , Manyu Li, Kathleen M. Caron
Kimberly L. Fritz-Six, … , Manyu Li, Kathleen M. Caron
Published December 20, 2007
Citation Information: J Clin Invest. 2008;118(1):40-50. https://doi.org/10.1172/JCI33302.
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Adrenomedullin signaling is necessary for murine lymphatic vascular development

Abstract

The lymphatic vascular system mediates fluid homeostasis, immune defense, and tumor metastasis. Only a handful of genes are known to affect the development of the lymphatic vasculature, and even fewer represent therapeutic targets for lymphatic diseases. Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through the calcitonin receptor–like receptor (calcrl) when the receptor is associated with a receptor activity–modifying protein (RAMP2). Here we report on the involvement of these genes in lymphangiogenesis. AM-, calcrl-, or RAMP2-null mice died mid-gestation after development of interstitial lymphedema. This conserved phenotype provided in vivo evidence that these components were required for AM signaling during embryogenesis. A conditional knockout line with loss of calcrl in endothelial cells confirmed an essential role for AM signaling in vascular development. Loss of AM signaling resulted in abnormal jugular lymphatic vessels due to reduction in lymphatic endothelial cell proliferation. Furthermore, AM caused enhanced activation of ERK signaling in human lymphatic versus blood endothelial cells, likely due to induction of CALCRL gene expression by the lymphatic transcriptional regulator Prox1. Collectively, our studies identify a class of genes involved in lymphangiogenesis that represent a pharmacologically tractable system for the treatment of lymphedema or inhibition of tumor metastasis.

Authors

Kimberly L. Fritz-Six, William P. Dunworth, Manyu Li, Kathleen M. Caron

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Figure 7

AM signaling is required for the proliferation and growth of jugular lymphatic vessels during embryonic development.

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AM signaling is required for the proliferation and growth of jugular lym...
(A–C) The percentage of proliferating cells relative to total cells was determined in the jugular vein and neighboring jugular lymph sac of wild-type (gray bars) and calcrl–/–, AM–/–, and RAMP2–/– (black bars, A–C, respectively) littermate embryos. Percent proliferative cells was defined as the number of BrdU-positive endothelial cells divided by the total number of endothelial cells in each vessel. Error bars indicate SEM. *P = 0.01 for calcrl–/– (A), P = 0.004 for AM–/– (B), and P = 0.01 for RAMP2–/– (C); Student’s t test. n > 6 animals per genotype. (D and E) Whole mount immunofluorescence of developing vasculature identified by PECAM staining and visualized by 3D OPT of wild-type (D) and RAMP2–/– (E) littermate embryos at E13.5. (F and G) Whole mount immunofluorescence of developing lymphatic vasculature identified by VEGFR3 staining and visualized by 3D OPT of wild-type (F) and RAMP2–/– (G) littermate embryos at E14.5. Compare the presence of well-formed jugular lymph sacs in the wild-type embryo (yellow arrows in F) with the relative lack of jugular lymphatic vessels in the RAMP2–/– littermate (G). Note that the retroperitoneal lymph vessel (red arrows) and dermal lymphatic vessels (green asterisks) of RAMP2–/– mice appear normal compared with those of wild-type embryos. Three embryos from 2 different litters were stained for PECAM and VEGFR3. The online supplemental data contains movies for enhanced, high-resolution, 3D viewing (Supplemental Figures 4 and 5). Scale bar: 2,000 μM.