Shane P. Herbert

Molecular control of endothelial cell behaviour during blood vessel morphogenesis.

The vertebrate vasculature forms an extensive branched network of blood vessels that supplies tissues with nutrients and oxygen. During vascular development, coordinated control of endothelial cell behaviour at the levels of cell migration, proliferation, polarity, differentiation and cell–cell communication is critical for functional blood vessel morphogenesis. Recent data uncover elaborate transcriptional, post-transcriptional and post-translational mechanisms that fine-tune key signalling pathways (such as the vascular endothelial growth factor and Notch pathways) to control endothelial cell behaviour during blood vessel sprouting (angiogenesis). These emerging frameworks controlling angiogenesis provide unique insights into fundamental biological processes common to other systems, such as tissue branching morphogenesis, mechanotransduction and tubulogenesis.

Arterial-Venous Segregation by Selective Cell Sprouting: An Alternative Mode of Blood Vessel Formation

Making Split Decisions Development of the vertebrate vasculature has been thought to involve just two mechanisms of blood vessel formation. Herbert et al. (p. 294; see the Perspective by Benedito and Adams) identified a third mechanism in zebrafish in which two distinct, unconnected vessels can be derived from a single precursor vessel. Several vascular endothelial growth factors and signaling pathways, including ephrin and notch signaling, coordinated the sorting and segregation of a mixture of arterial and venous-fated precursor cells into distinct arterial and venous vessels. These findings provide a mechanistic framework for how mixed populations of cells can coordinate their behavior to segregate and form distinct blood vessels. An alternative developmental pathway for vertebrate vasculature segregates a precursor vessel into two separate vessels. Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

Ligand-Stimulated VEGFR2 Signaling is Regulated by Co-Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF‐A)‐induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2‐mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF‐A‐stimulated signaling and endothelial cell migration. Ligand‐stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT‐0 subunit caused differential effects on ligand‐stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF‐A‐induced intracellular signaling to c‐Akt and endothelial nitric oxide synthase (eNOS). VEGF‐A‐stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF‐A‐mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C‐dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF‐stimulated endothelial signaling and cell migration.

The Confluence-dependent Interaction of Cytosolic Phospholipase A2-α with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A2-α (cPLA2α) cleaves phospholipids in a Ca2+-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca2+-independent association of cPLA2α with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA2α and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA2α is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA2α and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA2α activity and endothelial cell prostaglandin generation.

Endothelial cell confluence regulates Weibel-Palade body formation

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.

Group VIA calcium-independent phospholipase A2 mediates endothelial cell S phase progression

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis.

Activation of Cytosolic Phospholipase A2-α as a Novel Mechanism Regulating Endothelial Cell Cycle Progression and Angiogenesis

Release of endothelial cells from contact-inhibition and cell cycle re-entry is required for the induction of new blood vessel formation by angiogenesis. Using a combination of chemical inhibition, loss of function, and gain of function approaches, we demonstrate that endothelial cell cycle re-entry, S phase progression, and subsequent angiogenic tubule formation are dependent upon the activity of cytosolic phospholipase A2-α (cPLA2α). Inhibition of cPLA2α activity and small interfering RNA (siRNA)-mediated knockdown of endogenous cPLA2α reduced endothelial cell proliferation. In the absence of cPLA2α activity, endothelial cells exhibited retarded progression from G1 through S phase, displayed reduced cyclin A/cdk2 expression, and generated less arachidonic acid. In quiescent endothelial cells, cPLA2α is inactivated upon its sequestration at the Golgi apparatus. Upon the stimulation of endothelial cell proliferation, activation of cPLA2α by release from the Golgi apparatus was critical to the induction of cyclin A expression and efficient cell cycle progression. Consequently, inhibition of cPLA2α was sufficient to block angiogenic tubule formation in vitro. Furthermore, the siRNA-mediated retardation of endothelial cell cycle re-entry and proliferation was reversed upon overexpression of an siRNA-resistant form of cPLA2α. Thus, activation of cPLA2α acts as a novel mechanism for the regulation of endothelial cell cycle re-entry, cell cycle progression, and angiogenesis.

Cytosolic phospholipase A2‐α and cyclooxygenase‐2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus

Cytosolic phospholipase A2‐α (cPLA2‐α) is a calcium‐activated enzyme that plays an important role in agonist‐induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2‐α in human EA.hy.926 endothelial cells following stimulation with the calcium‐mobilizing agonist, A23187. Relocation of cPLA2‐α was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2‐α with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2‐α occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2‐α interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2‐α was seen to partially colocalize specifically with the COX‐2 isoform at the ER‐resembling structures, but not with COX‐1. These studies suggest that cPLA2‐α and COX‐2 may function together at a distinct and novel compartment for eicosanoid signalling.

Asymmetric division coordinates collective cell migration in angiogenesis

The asymmetric division of stem or progenitor cells generates daughters with distinct fates and regulates cell diversity during tissue morphogenesis. However, roles for asymmetric division in other more dynamic morphogenetic processes, such as cell migration, have not previously been described. Here we combine zebrafish in vivo experimental and computational approaches to reveal that heterogeneity introduced by asymmetric division generates multicellular polarity that drives coordinated collective cell migration in angiogenesis. We find that asymmetric positioning of the mitotic spindle during endothelial tip cell division generates daughters of distinct size with discrete ‘tip’ or ‘stalk’ thresholds of pro-migratory Vegfr signalling. Consequently, post-mitotic Vegfr asymmetry drives Dll4/Notch-independent self-organization of daughters into leading tip or trailing stalk cells, and disruption of asymmetry randomizes daughter tip/stalk selection. Thus, asymmetric division seamlessly integrates cell proliferation with collective migration, and, as such, may facilitate growth of other collectively migrating tissues during development, regeneration and cancer invasion.

Determination of Endothelial Stalk versus Tip Cell Potential during Angiogenesis by H2.0-like Homeobox-1

Summary Tissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) [3]. In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues.