We show here that induced systemic deletion of Rasa1 from adult mice resulted in a spontaneous disorder of the lymphatic vascular system. Loss of RASA1 caused extensive lymphatic vessel hyperplasia that was a consequence of dysregulated LEC proliferation. In addition, loss of RASA1 caused a lymphatic vessel leakage defect associated with the development of chylothorax, chylous ascites, and death. These findings identified RASA1 as a critical regulator of the lymphatic vessel growth and function required for the integrity of the lymphatic vasculature in resting animals.
To determine whether lymphatic vessel phenotypes in induced RASA1-deficient mice were LEC intrinsic, we used a Prox1ert2cre
knockin line, in which expression of Ert2Cre is driven by the endogenous Prox1
). Reporter analysis confirmed that Ert2Cre was active predominantly in lymphatic vessels in this line. A recent report showed that Prox1 is also expressed in venous valves and that in a Prox1ert2cre
BAC transgenic mouse line, Ert2Cre is active in venous valve endothelial cells as well as lymphatics (29
). Conversely, we were unable to detect Cre-mediated recombination in the blood vascular system using the Prox1ert2cre
knockin line described herein. Potentially, this discrepancy could be explained by differences in the level of Ert2Cre expression between lines and/or differences in the TM administration protocol.
Loss of RASA1 specifically in LECs of adult mice was shown to be sufficient for the development of lymphatic vessel growth defects. Hyperplasia of lymphatic vessels in the skin and diaphragm was noted, although in the latter tissue, the extent of hyperplasia was less than that observed upon ubiquitous deletion of Rasa1. In contrast, chylothorax did not develop in adult LEC-specific RASA1-deficient mice. One interpretation of this finding is that development of chylothorax is LEC extrinsic. However, it is more likely that the absence of chylothorax can be explained by relatively weak activity of Prox1ert2cre versus Ubert2cre in lymphatic vessels. In the Prox1ert2cre line, Cre-mediated recombination was detected in a minority of LECs in initial and collecting lymphatics, whereas in the Ubert2cre line, Cre activity was detected in all LECs. Thus, in order for chylothorax to manifest, a threshold level of Rasa1 deletion may have to be surpassed. In support of this, Rasa1fl/flProx1ert2cre mice that had been induced with TM at E15 of development, which would be predicted to result in more efficient Rasa1 deletion in LECs, did develop chylothorax 2 weeks after birth.
The LEC-intrinsic nature of the hyperplastic lymphatic vessel phenotype, together with the finding that MAPK was constitutively active in RASA1-deficient LECs in situ, suggested a model in which abnormal spontaneous lymphatic vessel growth arises as a result of dysregulated LEC growth factor receptor signal transduction to the low ligand concentrations normally present in resting tissues. According to this model, in wild-type mice, these ligands would continually initiate activation of Ras in LECs. However, owing to the action of RASA1, this activated Ras would be quickly converted back to the inactive GDP-bound form. In contrast, in RASA1-deficient mice, activated Ras molecules would accumulate, which in turn would result in dysregulated LEC proliferation and survival and overgrowth of the lymphatic vascular system.
Analysis of purified LECs in vitro revealed that Ras signal transduction initiated by PDGFR, FGFR, and VEGFR-3, but not by VEGFR-1 or VEGFR-2, was dysregulated in the absence of RASA1. Specifically, PDGF and FGF stimulated slightly prolonged activation of MAPK in RASA1-deficient LECs. Moreover, whereas VEGF-C stimulated a monophasic activation of MAPK and AKT in control LECs, a biphasic activation of both kinase types was observed in RASA1-deficient LECs. In parallel with these findings, RASA1-deficient LECs showed increased proliferation and survival in response to stimulation with the combination of all 3 growth factors in vitro. RASA1 has previously been implicated in the regulation of FGFR- and PDGFR-induced Ras activation (30
). To our knowledge, a similar role for RASA1 as a regulator of VEGFR-3–induced Ras activation has not been reported. However, like the other 2 receptors, VEGFR-3 contains cytoplasmic domain tyrosine residues in canonical motifs that would allow recognition by RASA1 SH2 domains and thus recruitment of RASA1 to membranes during the course of receptor signaling. It is likely that dysregulated Ras signal transduction through VEGFR-3 is the dominant factor that drives lymphatic hyperplasia in induced RASA1-deficient mice. This would be consistent with the finding that anti–VEGFR-3 blockade almost completely inhibited lymphatic vessel hyperplasia in the chest region of induced RASA1-deficient mice and had a significant inhibitory effect on lymphatic vessel hyperplasia in the skin.
Spontaneous blood vessel abnormalities were not observed in adult TM-induced Rasa1fl/flUbert2cre
mice. This cannot be explained by ineffective deletion of Rasa1
in BECs, since in Cre reporter mice, the Ubert2cre
transgene was shown to be highly active in blood vessels. Moreover, as shown by Western blotting, in Rasa1fl/flUbert2cre
mice, RASA1 expression in BECs was essentially extinguished in BECs (Supplemental Figure 5). One reason for the lack of a spontaneous blood vessel abnormality after loss of RASA1 in adult mice may be a redundancy of RASA1 with other RasGAPs in BECs. In this regard, extended activation of the Ras-MAPK pathway in response to different growth factors was not observed in RASA1-deficient BECs as it was in RASA1-deficient LECs. A second basis for the lack of a spontaneous blood vessel phenotype may be that activation of Ras by itself is not sufficient to drive BEC proliferation. This is indicated by the finding that transgenic overexpression of Ras in LECs and BECs in mice results in the same development of lymphatic vessel hyperplasia, but not blood vessel abnormalities, as described here (10
Although a spontaneous blood vessel phenotype was not observed in adult induced RASA1-deficient mice, it is important to point out that RASA1 does play an important role in the blood vascular system in adults during pathological angiogenesis. Thus, microRNA-mediated loss of RASA1 is essential for blood vessel angiogenesis toward tumors (6
). RASA1 is also critical for blood vessel function during development. In RASA1-deficient embryos, there is a failure of normal blood vessel patterning in both the yolk sac and the embryo itself, which leads to embryonic death (5
). Furthermore, as shown here, this was consequent to loss of RASA1 specifically in BECs (Supplemental Figure 1). Whether blood vascular defects in RASA1-deficient embryos develop as a result of aberrant Ras activation in BECs is uncertain (32
). RASA1 has been shown to be required for directed cell movement, the impairment of which may instead underlie defective blood vessel patterning in RASA1-deficient embryos. Importantly, RASA1 is thought to regulate directed cell movement in a manner independent of its ability to regulate Ras, but rather dependent on physical interaction with p190 RhoGAP, which functions as a GAP for the Ras-related Rho small GTP-binding protein.
In humans with CM-AVM, mutations of the RASA1
gene do cause spontaneous blood vascular lesions in children and adults (7
). This could reflect a genuine species difference between humans and mice with regard to the role of RASA1 in regulation of the mature blood vasculature. However, in CM-AVM, RASA1 loss through second-hit mutation may occur during embryonic development. In contrast, in the induced RASA1-deficient mouse model described here, expression of RASA1 remained intact throughout embryonic development. Therefore, should loss of RASA1 during development be necessary for the appearance of spontaneous blood vascular abnormalities that present after birth, differences in the timing of RASA1 loss in CM-AVM and in the induced RASA1-deficient mouse model could account for the differences in phenotype.
Interestingly, chylothorax and chylous ascites have been observed in a small number of CM-AVM patients (9
). Because chylothorax and chylous ascites are rare in humans, a spontaneous lymphatic vessel phenotype resulting from loss of RASA1 expression appears to be conserved across species. It is possible that a larger number of CM-AVM patients show lymphatic abnormalities, such as hyperplasia, that may be revealed by noninvasive NIR fluorescence imaging (34
). Alternatively, an apparent lower incidence of lymphatic abnormalities in CM-AVM could be explained by a requirement for second-hit mutations in LECs in order for lymphatic vessel lesions to manifest. Such second-hit mutations would be expected to occur in only a minority of LECs. This contrasts with the induced RASA1-deficient mouse, in which the vast majority of LECs would lose expression of RASA1.
VE-cadherin–rich button-like junctions between LECs in initial lymphatics of the chest region were disrupted in induced RASA1-deficient mice. This finding provides a potential explanation for lymphatic vessel leakage in this model. Notably, both tissue edema and chylothorax develop in mice that overexpress Ras in LECs (10
). This suggests that the lymphatic vessel leakage defects that occur after RASA1 loss are consequent to aberrant Ras activation in LECs. However, how aberrant Ras activation relates to disruption of button-like junctions remains to be established.
Outside of the lymphatic vascular system, no significant abnormalities have been detected in any other tissue in induced RASA1-deficient mice. One exception is the immune system, where T cell development and survival is affected (36
). However, T cell phenotypes in induced RASA1-deficient mice and T cell–specific constitutive RASA1-deficient mice are relatively subtle, and which RasGAPs regulate antigen-induced Ras activation in mature T cells remains to be defined. Likewise, despite its broad pattern of expression, RASA1 does not appear to be a significant nonredundant regulator of Ras activation in other somatic cell types (including fibroblasts; Supplemental Figure 5). In particular, induced RASA1-deficient mice did not develop tumors in any tissue; thus, RASA1 did not function as an essential tumor suppressor.
In summary, we showed here that RASA1 functions as a nonredundant physiological negative regulator of lymphatic vessel growth that is required for the maintenance of normal lymphatic vessel architecture and functional integrity in adult animals. These findings have important implications for our understanding of mechanisms of lymphatic vessel growth and function in general. In addition, prior findings suggest that RASA1 may be a useful target for the manipulation of lymphatic vessel growth in disease situations (37
). For example, in lymphedema, local short-term inhibition of RASA1 expression could promote local lymphatic vessel growth, resulting in symptom alleviation. Conversely, in cancer, prevention of growth factor–mediated downregulation of RASA1 in LECs, which may be required for tumor-induced lymphangiogenesis, could result in reduced tumor metastasis. One possible way in which such manipulation could be achieved is through the use of RASA1-specific microRNAs and counterpart anti-microRNAs that have previously been described and shown to be effective in manipulation of blood vessel angiogenesis in vivo (6