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Vascular tube morphogenesis requires the establishment of endothelial cell (EC) apical-basal polarity in three-dimensional (3D) extracellular matrices. To date, there is little understanding of how EC polarity is controlled during these highly dynamic and rapid morphogenic events. We show that the microtubule tip complex proteins, end binding 1 (EB1), p150(Glued), and Clasp1, control human EC tube formation by (1) inducing microtubule assembly and asymmetric cytoskeletal polarization, whereby acetylated and detyrosinated tubulins distribute in a subapical membrane location and filamentous actin distributes basally; (2) increasing tubulin posttranslational modifications, including required acetylation events; and (3) regulating an EC lumen signaling cascade that involves membrane type 1 matrix metallopatrinase (MT1-MMP)-dependent proteolysis as well as Pak, Raf, and Erk kinases. Another regulator of this process is the microtubule stabilizing protein, tau, which binds p150(Glued) and similarly affects EC lumen formation by controlling the levels of acetylated and detyrosinated tubulins. Increased expression of the tubulin deacetylases, sirtuin 2, and histone deacetylase 6 (HDAC6), blocks EC tube formation and cytoskeletal polarization, while siRNA suppression of these deacetylases stimulates these events. Overall, this work reveals a fundamental role for microtubule tip complex proteins in coordinating microtubule assembly, posttranslational modifications including acetylation, and apical-basal cytoskeletal polarization to control the developing apical membrane surface during blood vessel tubulogenesis in 3D matrix environments.

Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.

Here, we demonstrate a novel, direct-acting, and synergistic role for 3 hematopoietic stem cell cytokines: stem cell factor, interleukin-3, and stromal derived factor-1α, in controlling human endothelial cell (EC) tube morphogenesis, sprouting, and pericyte-induced tube maturation under defined serum-free conditions in 3-dimensional matrices. Angiogenic cytokines such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) alone or VEGF/FGF combinations do not support these responses. In contrast, VEGF and FGF prime EC responses to hematopoietic cytokines via up-regulation of c-Kit, IL-3Rα, and C-X-C chemokine receptor type 4 from either human ECs or embryonic quail vessel explants. In support of these findings, EC Runx1 is demonstrated to be critical in coordinating vascular morphogenic responses by controlling hematopoietic cytokine receptor expression. Combined blockade of hematopoietic cytokines or their receptors in vivo leads to blockade of developmental vascularization in quail embryos manifested by vascular hemorrhage and disrupted vascular remodeling events in multiple tissue beds. This work demonstrates a unique role for hematopoietic stem cell cytokines in vascular tube morphogenesis and sprouting and further demonstrates a novel upstream priming role for VEGF and FGF to facilitate the action of promorphogenic hematopoietic cytokines.

We show that endothelial cell (EC)–generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, α5β1, α3β1, α6β1, and α1β1, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

In this issue of Blood , Nichol and colleagues make the interesting and important observation that EGFL7 negatively regulates Notch activity in endothelial cells (ECs) to control blood vessel development.[1][1] Previous work demonstrates that EGFL7 is expressed in a highly restricted manner in

Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)–lined tubes in 3-dimensional (3D) extracellular matrices to stimulate vascular maturation including basement membrane matrix assembly. Through the use of this serum-free, defined system, we demonstrate that pericyte motility within 3D collagen matrices is dependent on the copresence of ECs. Using either soluble receptor traps consisting of the extracellular ligand-binding domains of platelet-derived growth factor receptor β, epidermal growth factor receptor (EGFR), and ErbB4 receptors or blocking antibodies directed to platelet-derived growth factor (PDGF)–BB, or heparin-binding EGF-like growth factor (HB-EGF), we show that both of these EC-derived ligands are required to control pericyte motility, proliferation, and recruitment along the EC tube ablumenal surface. Blockade of pericyte recruitment causes a lack of basement membrane matrix deposition and, concomitantly, increased vessel widths. Combined inhibition of PDGF-BB and HB-EGF–induced signaling in quail embryos leads to reduced pericyte recruitment to EC tubes, decreased basement membrane matrix deposition, increased vessel widths, and vascular hemorrhage phenotypes in vivo, in support of our findings in vitro. In conclusion, we report a dual role for EC-derived PDGF-BB and HB-EGF in controlling pericyte recruitment to EC-lined tubes during developmental vascularization events.

Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.