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Key Points Vascular endothelial growth factor receptor (VEGFR) signalling is tightly regulated at different levels: ligand and receptor expression, the presence of co-receptors and accessory proteins (that is, neuropilins, proteoglycans and integrins, among others) and inactivating tyrosine phosphatases. Together, these control the rate of cellular uptake, degradation and recycling. Canonical versus non-canonical signalling indicates VEGF-dependent versus non-VEGF-dependent activation of VEGFR2. Among the latter are mechanical forces, gremlins, galectins, lactate and low-density lipoprotein (LDL) cholesterol. VEGFR signalling output is regulated by crosstalk with numerous receptor systems, including fibroblast growth factor receptor (FGFR), AXL, Delta–Notch and Hippo pathways. VEGFR2 endocytosis and subsequent cytoplasmic trafficking have a key role in regulation of ERK signalling, which is crucial for numerous VEGF biological activities, including arterial fate determination, proliferation and migration. VEGF-dependent regulation of permeability involves T cell-soecific adapter (TSAd) and junctional SRC activation and crosstalk with AXL-dependent PI3K activation. Protein tyrosine phosphatases have important roles in regulation of specific VEGFR2-activated intracellular signalling events. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are crucial for the formation and remodelling of blood vessels. VEGFR2, which is the main endothelial VEGFR, is regulated by receptor-interacting proteins, endocytosis and trafficking. Recent insights have been gained into these layers of regulation and the crosstalk between VEGFR2 signalling and other endothelial signalling cascades. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are uniquely required to balance the formation of new blood vessels with the maintenance and remodelling of existing ones, during development and in adult tissues. Recent advances have greatly expanded our understanding of the tight and multi-level regulation of VEGFR2 signalling, which is the primary focus of this Review. Important insights have been gained into the regulatory roles of VEGFR-interacting proteins (such as neuropilins, proteoglycans, integrins and protein tyrosine phosphatases); the dynamics of VEGFR2 endocytosis, trafficking and signalling; and the crosstalk between VEGF-induced signalling and other endothelial signalling cascades. A clear understanding of this multifaceted signalling web is key to successful therapeutic suppression or stimulation of vascular growth.

Key Points The highly conserved Notch cell–cell signalling pathway operates in many different contexts across which the consequences can differ widely, despite the fact that the core pathway is very simple. Many different types of regulation contribute to the differing outcomes of Notch signalling, ranging from tissue-level coordination to nuclear governance. The pattern of expression of the ligands (which are transmembrane proteins), receptors and crucial modifying enzymes is one level of regulation that is common to many signalling pathways. However, the one-to-one interaction between ligand and receptor in Notch signalling places extra emphasis on this type of regulation, especially because the ligand and receptor can cis -inhibit one another when present in the same cells. 'Topological' tissue organization and the extent of cell–cell contacts are likely to be of unusual importance in influencing the levels of Notch activation because the ligands are transmembrane proteins. Nuclear context, in the form of cell-type-specific transcription factors and chromatin organization, is a primary level of control in generating qualitatively different outcomes after Notch activation. In addition, the wiring of the gene regulatory networks in the signal-receiving cells contributes to the diversity of responses and to the nature of its crosstalk with other signalling pathways. Together, these regulatory mechanisms make the Notch pathway versatile and able to undertake many different roles. But they are also susceptible to perturbations, and may be a contributory factor in Notch-related diseases. The Notch signalling pathway functions in many processes — from developmental patterning to cell growth and cell death. As the complexity of Notch signalling regulation is being unravelled at the levels of cell-surface ligand–receptor interactions and of gene expression, we are gaining a deeper understanding of how this conserved pathway can lead to such diverse cellular responses. The highly conserved Notch signalling pathway functions in many different developmental and homeostatic processes, which raises the question of how this pathway can achieve such diverse outcomes. With a direct route from the membrane to the nucleus, the Notch pathway has fewer opportunities for regulation than do many other signalling pathways, yet it generates exquisitely patterned structures, including sensory hair cells and branched arterial networks. More confusingly, its activity promotes tissue growth and cancers in some circumstances but cell death and tumour suppression in others. Many different regulatory mechanisms help to shape the activity of the Notch pathway, generating functional outputs that are appropriate for each context. These mechanisms include the receptor–ligand landscape, the tissue topology, the nuclear environment and the connectivity of the regulatory networks.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration 1 , 2 . This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2) 3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) ‘resets’ these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens 4 , 5 . In three-dimensional matrices—which do not have the constraints of bioprinted scaffolds—the ‘reset’ vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call ‘Organ-On-VascularNet’. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting. The transient reactivation of ETV2 in adult human endothelial cells reprograms these cells to become adaptable vasculogenic endothelia that in three-dimensional matrices self-assemble into vascular networks that can transport blood and physiologically arborize organoids and decellularized tissues.

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell–matrix signalling interactions. Loss of endothelial integrin β1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system. Langen et al.  identify a third capillary endothelial cell subtype, termed type E, that supports embryonic and early postnatal bone formation, and show that endothelial integrin β1 and laminin α5 are required for bone angiogenesis and osteogenesis.

The small intestinal villus tip is the first point of contact for lumen-derived substances including nutrients and microbial products. Electron microscopy studies from the early 1970s uncovered unusual spatial organization of small intestinal villus tip blood vessels: their exterior, epithelial-facing side is fenestrated, while the side facing the villus stroma is non-fenestrated, covered by pericytes and harbors endothelial nuclei. Such organization optimizes the absorption process, however the molecular mechanisms maintaining this highly specialized structure remain unclear. Here we report that perivascular LGR5 +  villus tip telocytes (VTTs) are necessary for maintenance of villus tip endothelial cell polarization and fenestration by sequestering VEGFA signaling. Mechanistically, unique VTT expression of the protease ADAMTS18 is necessary for VEGFA signaling sequestration through limiting fibronectin accumulation. Therefore, we propose a model in which LGR5 +  ADAMTS18 + telocytes are necessary to maintain a “just-right” level and location of VEGFA signaling in intestinal villus blood vasculature to ensure on one hand the presence of sufficient endothelial fenestrae, while avoiding excessive leakiness of the vessels and destabilization of villus tip epithelial structures. The molecular mechanisms ensuring the specialized structure of small intestinal villus tip blood vessels are incompletely understood. Here the authors show that ADAMTS18 +  telocytes maintain a “just-right” level and location of VEGFA signaling on intestinal villus blood vessels, thereby ensuring the presence of endothelial fenestrae for nutrient absorption, while avoiding excessive leakiness and destabilization of villus tip epithelial structures.

Angiogenesis is regulated by the dynamic interaction between endothelial cells (ECs). Hippo-Yes-associated protein (YAP) signalling has emerged as a key pathway that controls organ size and tissue growth by mediating cell contact inhibition. However, the role of YAP in EC has not been defined yet. Here, we show expression of YAP in the developing front of mouse retinal vessels. YAP subcellular localization, phosphorylation and activity are regulated by VE-cadherin-mediated-EC contacts. This VE-cadherin-dependent YAP phosphorylation requires phosphoinositide 3-kinase-Akt activation. We further identify angiopoietin-2 (ANG-2) as a potential transcriptional target of YAP in regulating angiogenic activity of EC in vitro and in vivo . Overexpression of YAP-active form in EC enhances angiogenic sprouting, and this effect is blocked by ANG-2 depletion or soluble Tie-2 treatment. These findings implicate YAP as a critical regulator in angiogenesis and provide new insights into the mechanism coordinating junctional stability and angiogenic activation of ECs. Angiogenesis is regulated by dynamic changes in endothelial cell contact. Here, the authors show that signals from endothelial cell junctions affect the subcellular localization and function of Yes-associated protein, ultimately modifying angiopoietin-2 expression and angiogenic activity of endothelial cells.

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial–venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs. Two studies by Sugden et al.  and Jin et al.  show that endoglin regulates endothelial cell migration through VEGFR2 signalling and controls blood vessel diameter in response to blood flow.

Loss-of-function (LOF) mutations in the endothelial cell (EC)-enriched gene endoglin ( ENG) cause the human disease hereditary haemorrhagic telangiectasia-1, characterized by vascular malformations promoted by vascular endothelial growth factor A (VEGFA). How ENG deficiency alters EC behaviour to trigger these anomalies is not understood. Mosaic ENG deletion in the postnatal mouse rendered Eng LOF ECs insensitive to flow-mediated venous to arterial migration. Eng LOF ECs retained within arterioles acquired venous characteristics and secondary ENG-independent proliferation resulting in arteriovenous malformation (AVM). Analysis following simultaneous Eng LOF and overexpression (OE) revealed that ENG OE ECs dominate tip-cell positions and home preferentially to arteries. ENG knockdown altered VEGFA-mediated VEGFR2 kinetics and promoted AKT signalling. Blockage of PI(3)K/AKT partly normalized flow-directed migration of ENG LOF ECs in vitro and reduced the severity of AVM in vivo . This demonstrates the requirement of ENG in flow-mediated migration and modulation of VEGFR2 signalling in vascular patterning. Two studies by Sugden et al. and Jin et al. show that endoglin regulates endothelial cell migration through VEGFR2 signalling and controls blood vessel diameter in response to blood flow.

Liver regeneration signals There is growing evidence to suggest that endothelial cells are not simply passive conduits for delivering oxygen and nutrients. During embryogenesis, for instance, they induce organogenesis before the circulation has developed. Experiments in a 70% partial hepatectomy liver regeneration model in mice now reveal a molecular pathway by which endothelial cells can sustain liver regeneration after surgical resection. VEGFR2 activation in a defined subpopulation of liver endothelial cells leads to the upregulation of the endothelial-specific transcription factor Id1, which in turn induces the secretion of Wnt2 and hepatocyte growth factor (HGF), which trigger hepatocyte proliferation. This suggests that vascular niche-derived inductive signals that promote liver regeneration could be utilized to initiate and accelerate liver recovery after these surgical procedures. These authors describe a molecular pathway by which endothelial cells sustain liver regeneration after surgical resection. Activation of vascular endothelial growth factor-A receptor-2 in a defined subpopulation of liver endothelial cells leads to the upregulation of the endothelial-specific transcription factor Id1 , which in turn induces Wnt2 and hepatocyte growth factor, which are secreted from the endothelial cells and trigger hepatocyte proliferation. During embryogenesis, endothelial cells induce organogenesis before the development of circulation 1 , 2 , 3 , 4 . These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis 5 , 6 , 7 . However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3 + CD34 − VEGFR2 + VE-cadherin + FactorVIII + CD45 − endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1–3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4–8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1 . Accordingly, Id1 -deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1 +/+ or Id1 −/− LSECs transduced with Wnt2 and HGF ( Id1 −/− Wnt2 + HGF + LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1 −/− mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2 + Id1 + Wnt2 + HGF + LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 ( tmem33 ), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33 -mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development. Calcium signalling downstream of VEGF is essential for VEGF-induced angiogenesis. Here Savage et al. show that Transmembrane Protein 33 (TMEM33) is required for angiogenesis and the endothelial calcium response to VEGF, revealing a function for TMEM33 in multicellular organisms.