NRP1 Regulates CDC42 Activation to Promote Filopodia Formation in Endothelial Tip Cells

摘要

class="head no_bottom_margin" id="sec1title">IntroductionDeveloping organs, ischemic tissues, and growing tumors produce the vascular endothelial growth factor VEGF-A to signal to its receptors on the endothelial cells (ECs) that line all blood vessels, and the resulting angiogenic expansion of local vasculature ensures the delivery of oxygen and nutrients to sustain fundamental metabolic processes (). VEGF-A signaling induces both the expansion of the EC pool by proliferation and the migration of ECs away from the existing plexus, whereby newly formed vessel sprouts are led by specialized tip cells that subsequently fuse to enable the formation of new vascular circuits (). The highly polarized endothelial tip cells can be distinguished from neighboring stalk cells by clusters of numerous long filopodia that are thought to detect microenvironmental cues for directional migration (). Filopodia are highly dynamic cellular protrusions that contain parallel bundles of filamentous actin (F-actin) and can extend from lamellipodia (). In addition to sensing growth factors, filopodia can adhere to the extracellular matrix (ECM) and form focal contacts that link the cytoskeleton to the ECM to promote forward movement.The main regulators of filopodia formation are members of the RHO-GTPase family, in particular CDC42, which is activated by VEGF-A signaling in cultured ECs (). Agreeing with a role for CDC42 in endothelial actin dynamics, both general and endothelial-specific CDC42 deletions disrupt blood vessel formation at the stage of vasculogenesis during mouse development (). However, the resulting early embryonic lethality of these mutants has precluded investigations into the role of CDC42 in filopodia formation, tip cell function, and sprouting angiogenesis in vivo. Moreover, it is not known if VEGF-A and/or ECM cues are important for CDC42 regulation during vessel sprouting.Neuropilin 1 (NRP1) is a non-tyrosine kinase transmembrane protein that regulates vascular development through dual roles in endothelial VEGF-A and ECM signaling (). Using the mouse embryo hindbrain as a model to study physiological angiogenesis, we recently demonstrated a cell-autonomous requirement for NRP1 in endothelial tip cells during angiogenic sprouting (). However, the specific cellular and molecular mechanisms that depend on NRP1 in tip cells have remained undefined. The prevailing model suggests that NRP1 acts as a VEGFR2 co-receptor downstream of VEGF-A signaling, which is chemotactic and induces the expression of essential tip cell genes. Supporting this idea, NRP1 can interact with VEGFR2 in ECs in vitro to potentiate VEGF-A signaling (e.g., ), and tip cell identity is promoted by VEGF-A signaling through VEGFR2 (). Alternatively, NRP1 may modulate signal transduction pathways that directly regulate tip cell behavior, such as cytoskeletal remodeling and filopodia extension. In agreement, NRP1 regulates filopodia orientation in hindbrain blood vessels () and enables actin remodeling for EC migration via ABL kinases (). However, it is not known how NRP1 might control filopodia formation and tip cell behavior.Here, we have combined the analysis of vascular development in the mouse hindbrain with functional studies in primary human ECs, zebrafish embryos, and mouse retina to demonstrate that NRP1 is dispensable for the genetic specification of tip cells but essential for CDC42 activation. Unexpectedly, NRP1 enabled CDC42-dependent actin remodeling and filopodia formation in endothelial tip cells independently of VEGF-A stimulation, and loss of CDC42 activation did not phenocopy the vascular defects of mice with impaired VEGF-A signaling through NRP1. Instead, NRP1-mediated CDC42 activation was induced by stimulation with ECM, and loss of this pathway caused defective vessel sprouting and branching similar to loss of the ECM-induced, NRP1-dependent activation of ABL kinases. In addition to demonstrating a physiological role for ECM-induced CDC42 activation in sprouting angiogenesis, our work has therefore identified a mechanism that ensures the integration of growth factor signals with ECM cues via NRP1 to enable tip cell function in sprouting angiogenesis.