RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice
Philip E. Lapinski, … , Eva Sevick-Muraca, Philip D. King
Philip E. Lapinski, … , Eva Sevick-Muraca, Philip D. King
Published January 9, 2012
Citation Information: J Clin Invest. 2012;122(2):733-747. https://doi.org/10.1172/JCI46116.
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Research Article Angiogenesis

RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice

Abstract

RASA1 (also known as p120 RasGAP) is a Ras GTPase–activating protein that functions as a regulator of blood vessel growth in adult mice and humans. In humans, RASA1 mutations cause capillary malformation–arteriovenous malformation (CM-AVM); whether it also functions as a regulator of the lymphatic vasculature is unknown. We investigated this issue using mice in which Rasa1 could be inducibly deleted by administration of tamoxifen. Systemic loss of RASA1 resulted in a lymphatic vessel disorder characterized by extensive lymphatic vessel hyperplasia and leakage and early lethality caused by chylothorax (lymphatic fluid accumulation in the pleural cavity). Lymphatic vessel hyperplasia was a consequence of increased proliferation of lymphatic endothelial cells (LECs) and was also observed in mice in which induced deletion of Rasa1 was restricted to LECs. RASA1-deficient LECs showed evidence of constitutive activation of Ras in situ. Furthermore, in isolated RASA1-deficient LECs, activation of the Ras signaling pathway was prolonged and cellular proliferation was enhanced after ligand binding to different growth factor receptors, including VEGFR-3. Blockade of VEGFR-3 was sufficient to inhibit the development of lymphatic vessel hyperplasia after loss of RASA1 in vivo. These findings reveal a role for RASA1 as a physiological negative regulator of LEC growth that maintains the lymphatic vasculature in a quiescent functional state through its ability to inhibit Ras signal transduction initiated through LEC-expressed growth factor receptors such as VEGFR-3.

Authors

Philip E. Lapinski, Sunkuk Kwon, Beth A. Lubeck, John E. Wilkinson, R. Sathish Srinivasan, Eva Sevick-Muraca, Philip D. King

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Figure 3

Lymphatic vessel dilation and hyperplasia in induced RASA1-deficient mice.

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Lymphatic vessel dilation and hyperplasia in induced RASA1-deficient mic...
(A and B) Anti–LYVE-1 antibody–stained sections of the chest wall of Rasa1fl/flUbert2cre and Rasa1fl/fl mice treated with TM 4 months prior (n = 25 per genotype). (A) Arrows denote representative lymphatic vessels within the chest wall. (B) Lower-power composite images. (C) Evans blue was injected i.d. at the base of the tail of shaved Rasa1fl/flUbert2cre and Rasa1fl/fl mice treated with TM 4 months prior. The dermal lymphatic network draining the injection site (IS) was imaged after 1 minute (top). After 5 minutes, a midline incision was made to expose lymphatic vessels and the inguinal LN (ILN) on the underside of the skin (bottom). The experiment was repeated 3 times with the same findings. (D) Representative anti–LYVE-1 antibody–stained sections of skin and lung of Rasa1fl/flUbert2cre and Rasa1fl/fl mice (n = 6 per genotype) treated with TM 3 months prior. Arrows denote lymphatic vessels. (E) Representative whole mount anti-Prox1 antibody staining of diaphragms of Rasa1fl/flUbert2cre and Rasa1fl/fl mice treated with TM 4 months prior (n = 5 per genotype). (F) Number of lymphatic vessels (LV) or Prox1+ cells per mm2 in skin and diaphragms, respectively, of Rasa1fl/flUbert2cre and Rasa1fl/fl mice in D and E. Data (mean + 1 SEM) were derived from 6 randomly selected fields from 3 mice per genotype. *P < 0.05; ***P < 0.001. Original magnification, ×400 (A); ×40 (B); ×100 (D and E).