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.
View: Text | PDF
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

×

Figure 9

Dysregulated Ras signal transduction in RASA1-deficient LECs.

Options: View larger image (or click on image) Download as PowerPoint
Dysregulated Ras signal transduction in RASA1-deficient LECs. (A) Serial...
(A) Serial sections of the chest wall of Rasa1fl/flUbert2cre and Rasa1fl/fl mice treated with TM 4 months prior were stained with anti–LYVE-1 or anti–phospho-ERK antibody. Arrows denote lymphatic vessels. Images are representative of 4 examined mice per genotype. Original magnification, ×100. (B) LECs were isolated from lungs of Rasa1fl/flUbert2cre and Rasa1fl/fl mice treated with TM 2 weeks prior (see Supplemental Figure 4). LECs were stimulated in vitro with the indicated growth factors for the indicated times. Activation of ERKs and AKT was determined by Western blotting of cell lysates with phosphospecific antibodies. Blots were reprobed with ERK and AKT antibodies to demonstrate equivalent protein loading. Experiments were repeated at least 3 times with the same results. (C) Purified LECs were stimulated with medium alone or with medium containing the indicated growth factors for 48 hours. Proliferation was assessed by BrdU incorporation during the last 24 hours of culture and measured by BrdU ELISA kit. Results are mean + 1 SD of triplicate determinations. (D) Purified LECs were grown to confluency and then cultured in the presence or absence of serum with ECGS for a further 72 hours. Apoptosis was determined by annexin V staining and flow cytometry. Numbers denote percent annexin V+ cells among total live 7-AAD– cells. Experiments in C and D were repeated 3 times with similar results.