Novel insights into the differential functions of Notch ligands in vascular formation
Journal of Angiogenesis Research. 2009;
Received: 19 August 2009 | Accepted: 16 November 2009 | Published: 16 November 2009
Vascular Cell ISSN: 2045-824X
The Notch signaling pathway is a critical component of vascular formation and morphogenesis in both development and disease. Compelling evidence indicates that Notch signaling is required for the induction of arterial-cell fate during development and for the selection of endothelial tip and stalk cells during sprouting angiogenesis. In mammals, two of the four Notch receptors (Notch1 and Notch4) and three of the five Notch ligands (Jagged1, Dll1, and Dll4) are predominantly expressed in vascular endothelial cells and are important for many aspects of vascular biology. During arterial cell-fate selection and angiogenesis, the roles of Notch1 and Notch4 are thought to be similar, and the function of Dll4 is well-characterized. However, the molecular mechanisms that determine the functional similarities and differences of Notch ligands in vascular endothelial cells remain largely unknown; consequently, additional research is needed to elucidate the ligand-specific functions and mechanisms associated with Notch activation in the vascular endothelium. Results from recent studies indicate that Dll1 and Dll4 have distinct roles in the specification and maintenance of arterial cell identity, while Dll4 and Jagged1 have opposing functions in tip- and stalk-cell selection during sprouting angiogenesis. This review will focus on the newly discovered, distinct functions of several Notch ligands in the regulation of blood vessel formation and will provide perspectives for future research in the field.
Notch signaling is evolutionarily conserved and critical for cell-fate determination, differentiation, and many other biological processes . The mammalian Notch signaling pathway is composed of four Notch receptors (Notch1-4) and five ligands (Jagged1 and 2 and Delta-like [Dll] 1, 3, and 4). All of the ligands are transmembrane-type proteins and, consequently, Notch signaling is often mediated by cell-cell interactions. Transmission generally occurs between neighboring cells that express high levels of either the receptor or the ligand, although receptor-ligand coexpression occurs in some cells, such as vascular endothelial cells. Over the last decade, numerous studies have demonstrated that Notch signaling is critically involved in vascular development and disease [2–6]. For example, Notch signaling is required for arterial cell-fate determination during embryonic development, and the Notch pathway controls both developmental and pathological angiogenesis by modulating the selection of endothelial tip and stalk cells in newly sprouting blood vessels. Regulation of the Notch pathway in blood vessels has been well characterized; however, the specific roles of each Notch ligand during vascular formation and morphogenesis are unknown. Recent studies provide insight into the distinct functions of Notch ligands in blood vessels, and this review summarizes the current understanding of how several ligands differentially activate Notch signaling in the vasculature.
Basic mechanisms of the Notch signaling pathway
Notch signaling is initiated by interactions between a Notch ligand expressed on the surface of one cell (the signaling cell) and a Notch receptor expressed on the surface of a neighboring cell (the receiving cell). Upon ligand binding, Notch is sequentially cleaved, and the Notch intracellular domain (NICD) is released into the cytoplasm. The NICD enters the nucleus, where it interacts with the transcription factor CSL (named after mammalian CBF1,
The extracellular domains of mammalian Notch ligands have several distinct features that participate in receptor binding (Figure 2). Their N-terminal regions contain a conserved module, and a second conserved module, the DSL (Delta/Serrate/LAG-2) domain, is located adjacent to the N-terminal region. Both Notch ligands and receptors contain multiple EGF-like repeats, and the ligands Jagged1, Jagged2, and Dll1 have tandem EGF repeats that form the DOS (Delta and OSM-11-like proteins) domain . Jagged1 and Jagged2 also contain a cysteine-rich domain located between the EGF-like repeats and the transmembrane domain. Both the DSL and DOS domains are critical for receptor binding , and the structural diversity of Notch ligands is determined by the presence or absence of the cysteine-rich DOS domains.
Activation of Notch signaling through cell-cell interactions (
Notch receptor and ligand expression in blood vessels
Notch1 is broadly expressed in many tissues, including the heart and vascular endothelial cells, while Notch4 expression is restricted to vascular endothelial cells [13–15], and Notch3 is predominantly expressed in vascular smooth muscle cells . Transcriptional regulation of
Notch1, Notch4, and the ligands Dll1 and Dll4 during arterial specification and maintenance
Results from recent studies in zebrafish suggest that activation of Notch signaling by the Sonic hedgehog (Shh) and VEGF pathways is essential for arterial specification during development [29, 30]. Two Notch receptors, Notch1 and Notch4, are predominantly expressed in arterial endothelial cells of early mouse embryos.
|Notch1||Proper vascular development; Postnatal neovascularization||[|
|Notch3||Maturation of vascular smooth muscle cells||[|
|Notch4||Null mice show normal vascular development; ||[|
|Jagged1||Dispensable for arterial specification; Formation of hematopoietic stem cells from the aorta; Smooth muscle differentiation and maturation; Proangiogenic regulation||[|
|Dll1||Maintenance of arterial identity; Arterial smooth muscle differentiation; Postnatal arteriogenesis||[|
|Dll4||Arterial specification; Tip cell and stalk cell selection during sprouting angiogenesis; Regulation of tumor angiogenesis||[|
Of the four Notch ligands (Jagged1, Jagged2, Dll1, and Dll4) that are expressed in arterial endothelial cells, Dll4 alone is expressed in the dorsal aorta of mice at embryonic day 8.5 (E8.5), and its expression is restricted to vascular endothelial cells ; thus, Dll4 is believed to be the ligand for Notch1 and Notch4 during early vascular development (Figure 3).
Dll1 expression is detected in arterial endothelial cells at a later stage (E13.5) of mouse development  and continues to be restricted to arterial endothelial cells in adults . Dll1 is not critically involved in arterial-cell specification; however, analyses in hypomorphic and endothelial-specific
Jagged1 does not play a critical role in arterial development [39–41] but is required for the definitive hematopoietic program in the dorsal aorta. After arterial and venous endothelial cells differentiate, the ventral region of the dorsal aorta, located in the aorta-gonad-mesonephros (AGM) region of the mid-gestation mouse embryo (around E10-11), generates the first adult hematopoietic stem cells (HSCs). Notch4 is broadly expressed throughout the aortic endothelium of the AGM, whereas Notch1 expression is restricted to the ventral region of the dorsal aorta [40, 42]. Importantly, three Notch ligands (Jagged1, Jagged2, and Dll4) have distinctive expression patterns in the dorsal aorta of the AGM: Jagged1 and Notch1 expression overlap in the dorsal aorta, Jagged2 expression occurs in endothelial cells adjacent to Notch1-positive endothelial cells, and Dll4 is expressed in both Notch1-positive and Notch1-negative endothelial cells [40, 42]. Analyses in
Notch3, Jagged1, and Dll1 during smooth-muscle differentiation and maturation
Notch3 is predominantly expressed in the vascular smooth muscle of arteries and is not expressed in veins. Mutations in human
Dll4 and Jagged1 in tip- and stalk-cell specification during sprouting angiogenesis
The formation of new blood vessels, a process known as angiogenesis, involves the sprouting of endothelial cells. In response to VEGF stimulation, filopodia extend from a migratory endothelial cell at the vessel's tip (i.e., the tip cell), and proliferative endothelial cells (i.e., stalk cells) form the trunk of the new vessel. Recent studies in mice and zebrafish clearly demonstrate that Notch signaling interacts with VEGF signaling during tip-cell and stalk-cell specification . VEGF induces Dll4 expression in tip cells, then Dll4 activates the Notch pathway in adjacent endothelial cells to reduce expression of VEGFR2 and VEGFR3, thereby suppressing the tip-cell phenotype, and tip-cell phenotype suppression cell-autonomously promotes the stalk-cell phenotype. Together, these mechanisms balance tip-cell and stalk-cell selection and, consequently, limit the number of sprouting vessels (Figure 7). Genetic or pharmacological disruption of Dll4-Notch signaling leads to excessive tip-cell formation and vessel sprouting in cultured cells, in zebrafish and mouse embryos, and during tumor angiogenesis [23, 25, 46–51].
By using endothelial-specific
Notch ligands in pathological angiogenesis
Dll4 is expressed in tumor vasculature [26, 36, 53, 54], and as observed in studies of developmental angiogenesis, the blockade of Dll4-mediated Notch signaling (via systemic administration of Dll4-neutralizing antibodies [47, 48] and systemic or local administration of modified Dll4 proteins [47, 55]) increased tumor-vessel sprouting, which indicates that Dll4-Notch signaling is critical for tip- and stalk-cell selection during tumor angiogenesis. Remarkably, the inhibition of Dll4-Notch signaling increased neovascularization but impaired tumor growth, because the non-productive angiogenesis reduced tumor perfusion. Conversely, Dll4 activation of endothelial Notch signaling reduces tumor angiogenesis, but increases vessel diameter and perfusion, which enhances tumor growth [47, 56]. For these reasons, Dll4 is now recognized as a potential therapeutic target for tumor angiogenesis .
As described above, Jagged1 antagonizes Dll4 during sprouting angiogenesis , and overexpression of Jagged1 in tumor cells has been shown to enhance neovascularization and tumor growth ; however, the role of Jagged1 in pathological angiogenesis (including tumor angiogenesis) is not yet fully understood. Current findings suggest that angiogenic sprouting in the tumor is tightly controlled by positive and negative regulation of Jagged1 and Dll4 in both endothelial and non-endothelial cells. Recent studies have shown that a soluble form of Notch1 (Notch decoy) acts as an antagonist of ligand-dependent Notch signaling by (potentially) interfering with Dll1, Dll4, and Jagged1 [59, 60]. Importantly, the Notch decoy reduces tumor growth without increasing vessel growth, which suggests that the effects of the Notch decoy differ from those induced by Dll4 blockade. It is therefore likely that the proangiogenic function of Jagged1 in tumor cells and endothelial cells could also influence tumor angiogenesis.
Notch signaling in peripheral ischemia
Notch signaling is also required for angiogenesis in peripheral ischemia models [32, 38] (Table 2). Blood flow recovery and postnatal neovascularization in response to hind-limb ischemia are impaired in both global and endothelial-specific
Concluding remarks and future perspectives
Studies performed in the past few years clearly demonstrate that the different Notch ligands have distinct functions in vascular development and disease. This understanding has prompted numerous investigations into the mechanisms by which Notch signaling is essential for multiple aspects of vascular biology. However, given that the effects of Notch pathway activation on endothelial cells are context-dependent , many questions remain to be answered. First, the upstream signaling pathways that control the expression of Notch ligands in blood vessels remain largely unknown; VEGF induces Dll4 expression in endothelial cells (Table 1), but Jagged1 is absent in tip cells where Dll4 is highly expressed, which suggests that the two ligands are regulated differently. Second, the selective activation of Notch in vascular endothelium remains unclear; for example, Notch signaling is not activated in arteries of
The author is an Associate Professor at Northwestern University School of Medicine, USA. He completed his postdoctoral training in the lab of Brigid Hogan at the Howard Hughes Medical Institute at Vanderbilt University Medical Center, USA. He graduated with a Ph.D. in Molecular and Cellular Biology from the University of Tokyo, Japan.
The author thanks W. Kevin Meisner, PhD, ELS, for editorial support. This work was supported by a NIH grant (RO1 HL074121) to TK.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
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