Here, we identify a critical requirement for Rasip1 during normal blood vessel tubulogenesis. We show that Rasip1 together with Arhgap29, a RhoA-specific GAP, suppresses RhoA signaling and dampens ROCK and non-muscle myosin IIA activities in endothelial cells (). When Rasip1 or Arhgap29 are absent, RhoA activation is elevated, resulting in excessive actomyosin contractility, disruption of cell polarity and consequent failure of lumen morphogenesis. Basal adhesion contacts, between ECs and surrounding ECM, fail to mature (as assessed by lack of integrin activation), resulting in disassociation of endothelium from surrounding mesenchyme. Apical adhesion is also disrupted, as junctional contacts are mislocalized within the central portion of cords, blocking the ability of ECs to open coherent lumens (). Inappropriate segregation of apicobasal character is evidenced by sustained overlap of apical and junctional (lateral) markers in Rasip1−/−
ECs. Additionally, Rasip1/Arhgap29 suppression of RhoA promotes Cdc42 and Rac1 activation, which in turn activates downstream kinases, including Pak4, Src, B-Raf, C-Raf and Erk, required for in vitro
EC lumen formation (Koh et al., 2008
; Koh et al., 2009
). Rasip1 thus represents a unique molecular bottleneck, as it integrates Rho family GTPase signaling to control cell polarity and adhesion, thereby coordinating endothelial morphogenesis and blood vessel tubulogenesis.
Models of Rasip1 regulation of embryonic vascular tubulogenesis
Rasip is required for endothelial cell polarity
Three key points led us to expect defective cell polarity in Rasip1
null angioblasts. First, Rasip1 regulates endothelial GTPase activity. Second, decades of classical work have demonstrated regulation of cell polarity by Rho GTPases, such as Cdc42 (Etienne-Manneville and Hall, 2002
). Finally, recent findings pointed to the importance of proper cell polarity during vascular lumen formation, both in early embryonic vessels, as well as those formed later during embryogenesis (Strilic et al., 2009
; Zovein et al., 2010
It was therefore surprising to observe no changes in subcellular localization or levels of CD34, F-actin, moesin or podocalyxin in Rasip1 null cord ECs, as these molecules are localized to the apical EC surface and are required for lumen formation. However, disruption of cell polarity in Rasip1 null cord ECs was indeed revealed by junctional protein mislocalization. Significant overlap of apical podocalyxin with ZO-1 in Rasip1 null ECs, and ectopic VE-cadherin, at multiple points along the apical surface and at all stages examined (both early and late), showed that the apical membrane did not properly segregate from the lateral/junctional membrane. Moreover, basal EC character was also compromised, as ectopic ZO-1 and VE-cadherin were found along basal membranes and functional integrin adhesion to the surrounding matrix was severely reduced.
Cell polarity defects in Rasip1
null cord ECs were further revealed by mislocalization of the polarity determinant Par3. Indeed, in the absence of Rasip1, we found a clear failure of Par3 to segregate normally to cell-cell junctions, away from the apical membrane. Par3 is known to associate with the aPKC polarity complex in epithelial cells (Suzuki and Ohno, 2006
) and to be important in establishment of arterial cell polarity (Zovein et al., 2010
). We note that while Par3 was previously found to be localized basally in older arterial vessels and reduced in the absence of β1 integrin (Zovein et al., 2010
), localization of Par3 in early angioblasts of the dorsal aortae was clearly junctional, and levels appeared unchanged in Rasip1
null ECs. It is conceivable that vasculogenic vessels form differently from angiogenic vessels formed later, but that key signaling molecules are nonetheless conserved.
We note that between clusters of junctional molecules (ZO-1/Par3/VE-cadherin), we observed slit-like openings, in which apical molecules like podocalyxin/moesin were expressed normally. We speculate that much of the lumen formation molecular pathway actually occurs ‘normally’ in the absence of Rasip1, albeit discontinuously. In Rasip1 null ECs, TJ/AJ localization likely occurs stochastically, such that the morphogenetic process is short-circuited, blocking functional lumen formation and tubulogenesis.
Rasip regulation of cell adhesion to ECM via maturation of adhesion contacts
The primary defect observed in ECs in the absence of Rasip1 function is failure of proper EC adhesion regulation, both between cells and with surrounding matrix. Failure of adhesion to matrix was evident both in live cell imaging of in vitro
EC lumen formation in siRasip1-treated cells, as well as in sections of Rasip1−/−
lumenless aortic cords. This EC-ECM failure was particularly striking in collagen 3D cultures, as ECs extended processes that transiently adhered to the surrounding matrix as they initiated lumen formation, but snapped back and collapsed repeatedly as they lost traction. These behavioral effects can be explained by impaired focal adhesion maturation in Rasip1-depleted cells. Whether resulting immature focal adhesions are merely weaker, or whether outside-in or inside-out signaling via focal adhesion components is compromised, can not be distinguishable from these studies. It will be interesting to identify additional Rasip1 binding partners, as it can complex with number Rho and Ras family GTPases (Mitin et al., 2004
), and further dissect signaling pathways and their effects on cell adhesion and architecture.
An interesting point regarding maturation of endothelial adhesion contacts is that Rasip1 appears to promote engagement of multiple integrins. Specifically, decrease of both β1 and αvβ3 in siRasip1-depleted cells, suggests other integrins are likely similarly regulated. Lumen dependence on multiple integrins is supported by antibody-based knockdowns targeting β1 (Drake et al., 1992
) or αvβ3 (Drake et al., 1995
), which blocked lumen formation in chick aortae. Redundancy of integrin dependence on Rasip1 during lumen formation likely explains the more profoundly compromised vascular phenotype observed in Rasip1−/−
mice versus EC-specific deletions of single integrins, which have not shown global lumen failure (Carlson et al., 2008
; Tanjore et al., 2008
). In Rasip1−/−
aortae, we suggest all required integrins have been blocked, as Rasip1 controls their activation. One relevant study bypassed the issue of integrin redundancy by examining fibronectin (FN) null embryos, as FN is a common ligand for a number of integrins, including β1 and αvβ3 (George et al., 1997
). Interestingly, the endocardial lumen was absent in FN−/−
embryos, and aortic ECs partially detached from surrounding mesenchyme, similar to Rasip1 null embryos. However, Rasip1−/−
do not phenocopy FN−/−
embryos, likely because other matrix components are also required. Together, these data suggest that Rasip1 is critical to integrin function in ECs.
Diverse cellular mechanisms drive vascular lumen formation?
An unexpected finding in our studies involved the growing gap between the trunk mesenchyme and the lumenless Rasip1−/− aortic cords. Our data suggests that Rasip1-deficient aortic ECs are unable to maintain proper integrin-mediated adhesion to the surrounding mesenchymal ECM and imply that the mesenchyme actively provides support during lumen expansion. We note that while this gap is pronounced surrounding the mutant aortic cords, similar gaps around other vessels are variable or absent, such as those in the yolk sac. It is therefore possible that different blood vessels create lumens by different cellular mechanisms, but common molecular mechanisms.
Indeed other biological ‘tubes’ often depend on multiple spatially- and/or temporally-specific mechanisms for lumen formation. The Drosophila trachea, for instance, ranges from intracellular vacuole-based lumen generation in fine terminal cells, to multicellular budding and lumen expansion in primary tracheal branches (Lubarsky and Krasnow, 2003
). Therefore, although we present here a mechanism for lumen formation based on angioblast adhesion regulation, we predict that the highly complex and heterogeneous developing vascular system (Aird, 2007
) will exhibit regional differences with respect to the cellular mechanisms employed to generate lumens.
Indeed, controversy has emerged in the field of vascular biology regarding how vascular lumens form. Some observations, both in vitro
and in vivo
, have suggested that lumen formation occurs via intracellular vacuoles, which emerge and fuse (Davis and Bayless, 2003
; Kamei et al., 2006
). While other observations have favored a mechanism based on angioblast cords rearranging their junctions to the periphery and opening up a central lumen based on active cytoskeletal forces (Jin et al., 2005
; Strilic et al., 2009
). Our in vivo
observations of murine aortic ECs reveal no evidence for vacuole fusion, similar to (Strilic et al., 2009
). However, cultured ECs in 3D matrices clearly exhibit vacuole fusion (seen in Movies S1
). Of great interest then is that failure of lumen formation in both systems occurs in the absence of Rasip1. This places Rasip1 in a critical regulatory position over the molecular machinery that controls cellular processes, such as the cell polarity, cytoskeleton (i.e. cell shape), and localization or robustness of cell junctions and adhesion contacts. Future studies will be aimed at identifying commonalities and differences in different EC types.
Rasip1 regulation of GTPase signaling
Our data suggests that Rasip1 suppresses RhoA (known to inhibit lumen formation) and promotes activation of Cdc42 and Rac1 (both known positively mediate lumen formation). Rescue experiments demonstrate that failure of tubulogenesis in the absence of either Rasip1, or its effector Arhgap29, can be fully restored by dominant negative RhoA or siRhoA treatment.
As a consequence, we have focused on Rasip1 regulation of RhoA signaling. However, the sharp decrease in Cdc42 and Rac1 activity in the absence of Rasip1 is of particular interest, as these GTPases play direct roles in assembly and disassembly of junctional components, as well as cytoarchitecture such as filopodia formation (Popoff and Geny, 2009
). Indeed, we see decreased filopodia in the absence of Rasip1 function (data not shown). In addition, Cdc42 has been directly linked to maintenance of EC-EC junctions via VE-cadherin (Broman et al., 2006
), as well as vacuole-based lumen formation (Bayless and Davis, 2002
). However, we identified no relevant effectors for Cdc42 or Rac1 as binding partners in our mass spectrometry screen, therefore it is possible that their regulation by Rasip1 is indirect and occurs via the RhoA pathway.
Critical role of actomyosin contractility for vascular tubulogenesis
One likely critical effector of vascular tubulogenesis is the RhoA-specific GAP and Rasip1 binding partner Arhgap29, which suppresses RhoA signaling specifically in developing blood vessels. Finding that Rasip1 also bound NMHCIIA, which is regulated by RhoA, raises the distinct possibility that a key role of Rasip1 is to suppress actomyosin contractility via suppression of RhoA. This is an appealing model, as angioblasts must dramatically change their shape during lumen formation, transforming from an initially rounded cuboidal morphology, to an exceedingly flattened one. Increased intracellular contractility would inhibit such a shape change. The recent report that NMHCIIA recruitment to the luminal surface of ECs was defective in the lumenless cords of VEGF heterozygotes (Strilic et al., 2009
) adds another interesting dimension to the role of contractility during lumen formation. In this case, lack of contractility fails to ‘bend’ the luminal membrane and ‘open up’ the central lumen. Together, the data suggests that precisely controlled levels of myosin II activity, and contractility, are required for vascular lumens: too much contractility (as in Rasip1−/−
ECs) or too little contractility (as in VEGF+/−
ECs) both lead to lumen failure.
Rasip1 therefore acts as a unique, endothelial-specific, integration node for Rho GTPase signaling, controlling EC morphogenesis from cord-to-patent blood vessel via cell polarity and adhesion regulation. Given that most approaches to anti-angiogenic therapies have focused on targeting extrinsic growth factors, such as VEGF, rather than cell intrinsic effects, future studies of Rasip1 and the molecular circuitry under its control, hold great promise to provide novel tools and models for furthering clinical therapies.