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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Research Article

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 2

Generation and characterization of conditional calcrl line.

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Generation and characterization of conditional calcrl line. ...
(A) Schematic diagram depicting strategy used for generation of a floxed calcrl allele by gene targeting. The top figure shows the endogenous wild-type calcrl allele. The targeting vector was designed so that loxP sites would flank the same exons that were deleted in the calcrl global knockout (36). The third line depicts the targeted calcrlFloxallele, and the fourth line depicts the calcrlLoxP allele after Cre-mediated excision. Primers used for isolation of correctly targeted ES cells and for routine genotyping are indicated by small arrows. (B) Correctly targeted ES cells were confirmed by Southern blot analysis using the probe depicted in A. (C) PCR genotyping for the calcrlFlox allele. (D) Quantitative RT-PCR was performed on RNA isolated from lungs and hearts of wild-type and homozygous calcrlFlox/Flox mice and revealed no significant differences in the expression of the calcrlFlox allele before Cre-mediated excision. (E) Schematic representation of breeding scheme used to generate mice with calcrl expression deleted specifically in endothelial cells by use of the Tie2Cre transgene. (F) Results of cross demonstrate that no viable calcrlLoxP/–Tie2Cre+mice were found beyond E16.5. (G) Compared with calcrlLoxP/+Tie2Cre+control littermates, the calcrlLoxP/–Tie2Cre+ mice displayed remarkable hydrops without hemorrhage, which phenocopied the global calcrl–knockout phenotype, yet often occurred substantially later, at E16.5. Original magnification, ×10.