Novel insights into the differential functions of Notch ligands in vascular formation

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.


Introduction
Notch signaling is evolutionarily conserved and critical for cell-fate determination, differentiation, and many other biological processes [1]. 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 transmembranetype 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][3][4][5][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 understand-ing 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, Drosophila Su(H), and Caenorhabditis elegans LAG1) to form a transcriptional activation complex that induces expression of the bHLH transcription factors (Hes and Hey families) ( Figure 1). This signaling mechanism is considered the "canonical" Notch pathway; non-canonical Notch signaling has also been reported [7].
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 [8]. 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 [9], 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 (trans-interactions) has been well characterized; however, Notch ligands also regulate the Notch pathway by binding to Notch receptors within the same cell (cisinteractions) [10,11]. In general, trans-interactions between Notch ligands and receptors activate Notch signaling, whereas cis-interactions are believed to inhibit Notch signaling [9]. The precise mechanisms that mediate Notch activation by the cis-interactions remain unclear, and further studies need to be performed [12].

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][14][15], and Notch3 is predominantly expressed in vascular smooth muscle cells [16]. Transcriptional regulation of A diagram of the canonical Notch signaling pathway Figure 1 A diagram of the canonical Notch signaling pathway. This schematic shows a simplified overview of the main components of Notch signaling. Upon Notch ligand binding, a two-step proteolysis cleavage process (small arrows) within the juxtamembrane region and transmembrane domain of the Notch receptor is catalyzed by a member of the disintegrin and metalloproteases (ADAMS) family and the γsecretase containing complex, respectively, then the Notch intracellular domain (NICD) is released from the membrane and translocates to the nucleus, where it forms a transcriptional activation complex with CSL and coactivators (CoA), thereby inducing the transcription of target genes. Five mammalian ligands are classified into two categories, Delta-like (Dll1, Dll3, Dll4) and Serrate-like (Jagged1, Jagged2), based on structural homology to the two Drosophila ligands, Delta and Serrate. All Notch ligands have an N-terminal domain, a DSL (Delta/Serrate/LAG-2) domain and EGF-like repeats. Jagged1 and Jagged2 contain a cysteinerich domain, whereas Jagged1, Jagged2, and Dll1 have two DOS (Delta and OSM-11-like proteins) domains located immediately following the DSL domain.

Domain organization of mammalian Notch ligands
Notch4 in vascular endothelial cells is controlled by fibroblast growth factor 2 (FGF2), the signal-dependent transcription-factor activator protein 1 (AP-1), and the glucocorticoid receptor [14,15,17]. Four of the five known mammalian Notch ligands (Jagged1, Jagged2, Dll1, and Dll4) are expressed in vascular endothelial cells [13,[18][19][20]; Jagged1 is also expressed in smooth muscle cells surrounding the arteries and plays an important role in smooth muscle cell maturation [21]. The molecular mechanisms that control the expression of Notch ligands in vascular endothelial cells and smooth muscle cells have been frequent topics of recent research (Table 1). For example, Dll4 expression during arterial specification and for tip-cell selection during vessel sprouting [22][23][24][25][26][27][28] is mediated by vascular endothelial growth factor (VEGF). Importantly, and as described below, the expression patterns of the Notch ligands vary both spatially and temporally and, consequently, the localization of each ligand is likely to be critical for Notch activation in blood vessels (Figures 3, 4, 5, 6, and 7).

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 mutant mice die with cardiovascular defects during early development [13], and endothelial-specific ablation of Notch1 in mice leads to embryonic lethality and vascular abnormalities that are associated with angiogenesis [31]. An endothelial-specific Notch1+/-mutation has also been associated with impaired postnatal neovascularization in a marine hind-limb ischemia model [32]. These results demonstrate the cell-type specific role of Notch1 in the vascular endothelium during development and postnatal life ( Table 2). Notch4 mutant mice display no conspicuous phenotype, but the vascular defects observed in compound Notch1; Notch4 mutant embryos are more severe than those in Notch1 mutants [13], which suggests that the functions of Notch1 and Notch4 overlap during vascular development. Endothelial expression of a constitutively active Notch4 mutant from the Flk1 (VEGFR2) locus results in embryonic vascular abnormalities such as disorganized vascular networks and dilated blood vessels [33], and in adult mice, constitutively active Notch4 expression has been associated with arteriovenous malformations (AVMs) (i.e., abnormal connections between arteries and veins) that are accompanied by ectopic expression of the arterial marker ephrinB2 in veins [34]. Furthermore, the Arterial cell specification mediated by Dll4-Notch signaling  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 [13]; thus, Dll4 is believed to be the ligand for Notch1 and Notch4 during early vascular develop-Opposing effects of Dll4 and Jagged1 on sprouting angiogen-esis Smooth-muscle maturation mediated by Jagged1/Dll1-Notch3 signaling Figure 6 Smooth-muscle maturation mediated by Jagged1/ Dll1-Notch3 signaling. Jagged1 and Dll1 in endothelial cells activate Notch3 on mural cells, thereby promoting mural-cell maturation. ment ( Figure 3). ; thus, the function of Jagged1 is distinct from Dll4 and Jagged2 activity during the hematopoietic program of the newly formed aorta ( Figure  5).

Notch3, Jagged1, and Dll1 during smoothmuscle differentiation and maturation
Notch3 is predominantly expressed in the vascular smooth muscle of arteries and is not expressed in veins.
Mutations in human NOTCH3 are associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a disorder that causes stroke and dementia and is accompanied by the degeneration of vascular smooth muscle cells [43]; adult Notch3 mutant mice display a defect in the maturation of arterial smooth muscle cells [44]. As noted above, Jagged1 mutant mice exhibit normal arterial development [39,40], yet endothelial-specific Jagged1 mutants have impaired vascular smooth muscle differentiation [39]. This observation indicates that Jagged1 expression in the arterial endothelium activates Notch in neighboring cells, and that this function is critical for smooth muscle cell differentiation. Jagged1 expression by endothelial cells induces mural cells (pericytes in the microvasculature or smooth muscle cells in larger vessels) to express Notch3 and Jagged1, which subsequently promotes and maintains the differentiation phenotype of mural cells [21], whereas platelet-derived growth factor (PDGF) and angiotensin II downregulate Notch3 and Jagged1 expression in vascular smooth muscle cells [45]. Furthermore, a recent study found that expression of the arterial smooth muscle marker smoothelin is impaired in Dll1 mutant mice [19], and this decline has also been observed in Notch3-mutant arteries [44]. Taken together, these findings suggest that Jagged1 and Dll1 are the primary ligands that regulate Notch3 activity during smooth-muscle differentiation and maturation ( Figure 6).

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 [5]. 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][47][48][49][50][51].
By using endothelial-specific Jagged1 mutant mice and mice that overexpress Jagged1 in vascular endothelial cells, Benedito et al. demonstrated that Jagged1 enhances angiogenesis and antagonizes the effects of Dll4-mediated Notch signaling during sprouting angiogenesis [52]. Jagged1 is strongly expressed in stalk cells, whereas Dll4 is predominantly detected in tip cells [52], and the antagonistic interaction between Dll4 and Jagged1 in endothelial cells is mediated by the glycosyltransferase Fringe, which regulates the posttranslational modifications of Notch receptors in a ligand-dependent manner. Fringe enhances Notch activation in response to Delta-like ligands and reduces Notch activity in response to Jagged ligands [12]; consequently, Fringe increases Dll4-induced endothelial Notch signaling and reduces Notch signaling in response to Jagged1 [52]. Jagged1 also appears to promote vascular sprouting by regulating VEGFR3 expression in tip cells [52]. Taken together, these results illustrate the opposing effects of Dll4 and Jagged1 on sprouting angiogenesis.

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 [57].
As described above, Jagged1 antagonizes Dll4 during sprouting angiogenesis [52], and overexpression of Jagged1 in tumor cells has been shown to enhance neovascularization and tumor growth [58]; however, the role of Jagged1 in pathological angiogenesis (including tumor angiogenesis) is not yet fully understood. Current find-ings 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 liganddependent 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] (

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 [4], 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 Dll1 mutant mice, despite the presence of Jagged1 and Dll4 [19]. Third, the role of noncanonical Notch ligands, such as microfibril-associated glycoprotein (MAGP)-2 [9], is poorly understood. MAGP-2 binds to Jagged1, Jagged2, Dll1, and Notch1 [61,62], and is known to modulate Notch signaling in sprouting angiogenesis [63,64], but the mechanistic basis for the function of MAGP-2 in ligand-dependent Notch activation has yet to be elucidated. Finally, given that Dll4 and Jagged1 have opposing effects on angiogenesis, experiments that specifically inhibit each ligand with selective neutralizing antibodies [65] may be important not only for understanding how Notch is activated in the vasculature, but also for the development of therapeutic strategies designed to control angiogenesis by targeting Notch signaling. Arterial specification; Tip cell and stalk cell selection during sprouting angiogenesis; Regulation of tumor angiogenesis [23,25,[35][36][37][46][47][48][49][50][51][52]55,56]