Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II

Abstract

Rationale Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells (ECs), apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 is required for apical junction clearance, as well as for regulation of Rho GTPase activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. Objective The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. Methods and Results Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of EC apical junctions during vascular tubulogenesis depends on Ras interacting protein 1 (Rasip1), as well as the GTPase Cdc42 and the kinase Pak4. Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all block EC tubulogenesis. By contrast, inactivation of RhoA signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII either prior to, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. Conclusions Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.

Afadin and RhoA control pancreatic endocrine mass via lumen morphogenesis

“…We note that Afadin is the epithelial counterpart of Rasip1, which we showed to be an endothelial-specific molecule required for continuous vascular lumen formation (Xu et al 2011). Our previous findings also suggest that Rasip1 is involved in vesicular trafficking (Barry et al 2016). Therefore, Afadin and Rasip1 may represent analogous molecules that carry out similar cellular functions to direct coordinated lumenogenesis in the epithelium and endothelium, respectively.…”

“…These lumens continue to develop via extension and interconnection of existing lumens. RhoA-directed actomyosin machinery is critical to the lumen extension process, and we showed recently that endothelial lumen formation relies on actomyosin activity (Lee and Kolodziej 2002;Denker et al 2015;Barry et al 2016). We thus hypothesized that lumen morphogenesis within the central epithelium would also require actomyosin contractility.…”

The biomechanical properties of an epithelial tissue determine the location of its vasculature

“…We also applied Y27632, a frequently applied inhibitor of Rho-associated protein kinase (ROCK)58, which was previously shown to be downstream of ILK3435. The levels of cortex tension were reduced by both treatments, and no longer significantly differed between control and ILK-depleted islet cells (Fig.…”

Rasip1 controls lymphatic vessel lumen maintenance by regulating endothelial cell junctions

“…Previous studies demonstrated that, in blood vessels, Rasip1 regulation of VE-cadherin-mediated EC-EC junctions and integrin-mediated EC-ECM junctions is crucial for blood vessel tubulogenesis and blood endothelial junctions (Xu et al, 2011). In these cells, Rasip1 inhibits RhoA, and in turns promotes the activity of Cdc42 and Rac1, to allow controlled expansion of vessel lumens during embryonic growth (Xu et al, 2011;Barry et al, 2016). To evaluate these regulatory pathways in LECs, we carried out an in vitro culture approach to remove RASIP1 function and found a significant reduction in CDC42 activity in siRNA-treated human dermal LECs, compared with controls ( Fig.…”

“…The cord-to-tube transition involves a series of cellular changes that are required for lumen initiation and lumen expansion, including junction molecule re-localization and distribution. Rasip1 is required in these processes by temporally regulating different pools of Rho GTPases to clear the adhesions from the apical membrane to form a single, continuous lumen (Xu et al, 2011;Barry et al, 2016) (Fig. 10A).…”