RASA1 regulates the function of lymphatic vessel valves in mice
Philip E. Lapinski, … , Michael J. Davis, Philip D. King
Philip E. Lapinski, … , Michael J. Davis, Philip D. King
Published May 22, 2017
Citation Information: J Clin Invest. 2017;127(7):2569-2585. https://doi.org/10.1172/JCI89607.
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Research Article Vascular biology

RASA1 regulates the function of lymphatic vessel valves in mice

Abstract

Capillary malformation–arteriovenous malformation (CM-AVM) is a blood and lymphatic vessel (LV) disorder that is caused by inherited inactivating mutations of the RASA1 gene, which encodes p120 RasGAP (RASA1), a negative regulator of the Ras small GTP-binding protein. How RASA1 mutations lead to the LV leakage defects that occur in CM-AVM is not understood. Here, we report that disruption of the Rasa1 gene in adult mice resulted in loss of LV endothelial cells (LECs) specifically from the leaflets of intraluminal valves in collecting LVs. As a result, valves were unable to prevent fluid backflow and the vessels were ineffective pumps. Furthermore, disruption of Rasa1 in midgestation resulted in LEC apoptosis in developing LV valves and consequently failed LV valvulogenesis. Similar phenotypes were observed in induced RASA1-deficient adult mice and embryos expressing a catalytically inactive RASA1R780Q mutation. Thus, RASA1 catalytic activity is essential for the function and development of LV valves. These data provide a partial explanation for LV leakage defects and potentially other LV abnormalities observed in CM-AVM.

Authors

Philip E. Lapinski, Beth A. Lubeck, Di Chen, Abbas Doosti, Scott D. Zawieja, Michael J. Davis, Philip D. King

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Figure 5

LV valve leaflet atrophy associated with reduced numbers of leaflet PROX1+ LECs in induced RASA1-deficient mice.

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LV valve leaflet atrophy associated with reduced numbers of leaflet PROX...
All analyses were performed with popliteal LV valves. (A) Still image of popliteal LV valve from a back-leak experiment indicating parameters measured for B–D. (B) Mean vessel diameter ± SEM at different upstream pressures (Pin) of LVs from Rasa1fl/fl (n = 4) and Rasa1fl/fl Ubert2cre (n = 7) mice treated with tamoxifen 9 weeks previously. (C) Mean sinus diameter + SEM at Pout 10 cm H2O of LVs from littermate Rasa1fl/fl and Rasa1fl/fl Ubert2cre mice treated with tamoxifen 1, 9, and 16 weeks previously (n = 6 of each genotype at 1 and 16 weeks; n = 3 Rasa1fl/fl and n = 7 Rasa1fl/fl Ubert2cre mice at 9 weeks after tamoxifen treatment). (D) Mean leaflet length + SEM of LVs from littermate Rasa1fl/fl and Rasa1fl/fl Ubert2cre mice treated with tamoxifen 1, 9, and 16 weeks previously (n = 12 of each genotype at 1 and 16 weeks; n = 6 Rasa1fl/fl and n = 13 Rasa1fl/fl Ubert2cre at 9 weeks after tamoxifen treatment). (E) Confocal microscopy images of popliteal LVs from littermate Rasa1fl/fl and Rasa1fl/fl Ubert2cre mice administered tamoxifen 5 weeks beforehand. Vessels were stained with integrin α-9 and PROX1 antibodies to highlight valve leaflets. Larger images at left show whole-valve regions. Smaller images at right show valve leaflets only from the same valves. One valve leaflet image is marked to indicate maximum leaflet depth, and outer perimeter is shown in F. (F) Graphs show maximum leaflet depth, leaflet outer perimeter, and number of PROX1+ cells per leaflet for different LV valve leaflets from Rasa1fl/fl and Rasa1fl/fl Ubert2cre mice administered tamoxifen 5 weeks beforehand. Mean + SEM are indicated. *P < 0.05; ****P < 0.0001, Student’s 2-sample t test.