RASA1 regulates the function of lymphatic vessel valves in mice
Philip E. Lapinski, Beth A. Lubeck, Di Chen, Abbas Doosti, Scott D. Zawieja, Michael J. Davis, Philip D. King
Philip E. Lapinski, Beth A. Lubeck, Di Chen, Abbas Doosti, Scott D. Zawieja, Michael J. Davis, Philip D. King
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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 6

Adult mice that are genetically altered to express an R780Q catalytically inactive form of RASA1 alone develop chylothorax, LV hyperplasia, and LV valve dysfunction.

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Adult mice that are genetically altered to express an R780Q catalyticall...
(A) Rasa1fl/R780Q Ubert2cre mice were administered tamoxifen at 3 weeks of age to disrupt the Rasa1fl allele, resulting in expression of the Rasa1R780Q allele alone. (B) Kaplan-Meier survival curves of littermate Rasa1fl/R780Q Ubert2cre, Rasa1fl/fl Ubert2cre, Rasa1fl/R780Q, and Rasa1fl/fl mice, all administered tamoxifen at 3 weeks of age (n = 12 mice of each genotype). (C) Flow cytometric analysis of pleural effusion cells from moribund tamoxifen-treated Rasa1fl/R780Q Ubert2cre and Rasa1fl/fl Ubert2cre mice showing expression of the lymphocytic markers TCRβ (T cells) and B220 (B cells). Numbers in plots denote the percentages of live cells. Results are representative of at least 4 mice examined of each genotype. (D) LYVE-1 antibody staining of the chest wall of moribund tamoxifen-treated Rasa1fl/R780Q Ubert2cre and Rasa1fl/fl Ubert2cre mice showing extensive LV hyperplasia (arrowheads) at the pleural cavity face. (E–G) Valve function tests were performed with popliteal LVs from littermate Rasa1fl/R780Q and Rasa1fl/R780Q Ubert2cre mice at the indicated times after tamoxifen treatment that was administered at 2 months of age. (E) Pump-function tests. Symbols represent individual valves. (F) Valve-closure tests. Within each graph, different colored symbols represent the same valve. (G) Low-pressure back-leak tests showing mean Psn + SEM at a Pout of 10 cm H2O. Rasa1fl/R780Q, n = 3 and n = 6 at 12 and 17–24 weeks, respectively; Rasa1fl/R780Q Ubert2cre, n = 3 at 5 and 12 weeks and n = 6 at 17–24 weeks. **P < 0.001, Student’s 2-sample t test. See also Supplemental Videos 8 and 9, which show examples of valves from vessels of Rasa1fl/R780Q Ubert2cre mice, 12 and 23 weeks after tamoxifen treatment respectively, in low pressure back-leak tests.