There is good evidence to indicate that members of the Notch family of transmembrane receptors play an important role in regulating cell fate decisions and differentiation (2
). More recently, several studies point to a role for Notch and its ligands in influencing vascular development. Mutations in Notch3 are responsible for the human vascular disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, although in this case the defect appears to be mainly in vascular smooth muscle cells (37
). Mutations in presenilin 1, a protein involved in Notch proteolytic processing, results in hemorrhaging (67
). Mice that are rendered null for Notch ligands Jagged1 and Delta1 exhibit vascular remodeling defects (80
) and hemorrhaging (32
), respectively. Antisense oligonucleotides directed against Jagged1 enhance FGF-2-induced endothelial tube formation in a collagen gel assay (81
). Recently, a study examining the expression of four Notch receptors (Notch1 to -4) and five Notch ligands (Delta1, -3, and -4 and Jagged1 and -2) in the developing mouse vasculature was performed. Notch1, Notch3, Notch4, Delta4, Jagged1, and Jagged2 are all expressed in arteries but not veins (76
). Notch2, Delta1, and Delta3, on the other hand, are not expressed in vessels (76
). Nevertheless, a Notch2 hypomorphic allele disrupts vessel remodeling in multiple vascular beds (50
). The combined loss of Notch4 and Notch1 functions due to gene targeting in the mouse results in defects in vascular remodeling (34
). Interestingly, expression of activated Notch4 in the mouse embryonic vasculature, under the control of the VEGF-R2 promoter, also results in vascular patterning defects (73
). Although the last two studies demonstrate that both increases and decreases in Notch4 signaling result in a common vascular phenotype, disrupted blood vessel development, a mechanism(s) by which to explain this phenotype has not been elucidated.
The enforced expression of a constitutively active form of murine Notch4 in a mammary epithelial cell line has been shown to inhibit branching morphogenesis in a collagen gel assay (75
). Because mammary epithelial tubulogenesis and blood vessel angiogenesis are similar morphogenic processes (52
) and because Notch4 is primarily expressed in the endothelium (47
), we investigated whether enforced expression of activated Notch4 (Notch4IC) in endothelial cells could inhibit endothelial sprouting in vitro and angiogenesis in vivo. In an in vitro endothelial-tube formation assay, we show that Notch4IC inhibits spontaneous endothelial sprouting, as well as sprouting in response to FGF-2 and VEGF (Fig. ). Furthermore, using an in vivo chick CAM assay, we demonstrate that Notch4IC expression is sufficient to inhibit VEGF-induced angiogenesis (Fig. and ).
Quiescent endothelial cells are normally anchored by their abluminal surface to a collagen-rich matrix (38
). At the initiation of angiogenesis, the mature collagen-containing matrix is degraded and replaced by a provisional matrix of fibrin and fibronectin upon which endothelial cells migrate and proliferate (20
). The endothelial-sprouting assay used in our studies mimics angiogenesis in vivo. Specifically, microvascular endothelial cells are seeded as a monolayer onto gelatin-coated beads and are then induced by angiogenic factors to migrate into a fibrin matrix to form sprouts. We report that endothelial cells expressing Notch4IC exhibit inhibited sprouting in vitro (Fig. and ) and that this inhibition can be explained in part by an increase in HMEC-Notch4IC adhesion to collagen (Fig. to ). By enhancing cell adherence to collagen-coated beads, activated Notch4 prevents migration of the cells into the fibrin matrix. This is in accordance with our migration studies, where HMEC-Notch4IC migration through collagen, but not fibrinogen, was inhibited (Fig. ). Proliferation rates, on the other hand, in HMEC-Notch4IC and control cells were found to be similar (Fig. ). Our in vivo studies demonstrate that Notch4IC expression in the chick CAM inhibits VEGF-induced angiogenesis (Fig. and ). Based on our in vitro findings, the inhibition of angiogenesis in vivo may be due in part to enhanced endothelial cell adhesion to matrix proteins, thereby inhibiting vascular remodeling in the CAM.
Cell migration requires the coordinated activation and deactivation of integrins (46
). As a cell migrates across a matrix, integrins at the leading edge of the cell adhere to the substrate (35
). At the same time, receptors at the trailing edge of the cell detach from the substrate to allow the cell to progress forward (56
). Thus, during the sprouting process of angiogenesis, integrin affinity states are constantly being modulated. The αvβ3- integrin has been shown to play a critical role in angiogenesis, but several studies also delineate the essential contribution of β1-integrins in endothelial morphogenesis (7
). Our data show that activated Notch4 increases endothelial cell adhesion (Fig. ) and that enhanced β1-integrin affinity plays a role in this increased adhesion (Fig. and ).
Our work demonstrates that constitutive Notch4 activation inhibits vascular remodeling. Importantly, our studies provide a possible mechanism with which to explain the common vascular defects observed in mutant mice with either increased (73
) or decreased (45
) Notch signaling. Because Notch plays a role in cell fate decisions, Notch signaling must be precisely regulated and hence requires cessation of receptor signaling at certain times (2
). Similarly, because cell adhesion influences cell functions such as migration and cell phenotype, modulation of cell adhesion must be strictly regulated (7
). Therefore, it is possible that knocking out Notch4 and Notch1 results in a loss of cell-to-extracellular matrix adhesion and hence inhibited vascular remodeling, whereas constitutive Notch4 activation results in excessive cell-to-extracellular matrix adhesion, thereby effectively fixing the cells in place. Taken together, our studies as well as the studies of Krebs et al. (45
) and Uyttendaele et al. (73
) reveal that altered Notch4 signaling results in disrupted blood vessel development.
Notch-like extracellular matrix protein Del1 has been shown to induce integrin signaling and angiogenesis by binding endothelial αvβ3 and promoting migration (58
). This is a case of signaling from the outside to the interior of the cell, as seen with many transmembrane receptors. In contrast, our studies suggest that activation of Notch4 propagates signals that induce an active, high-affinity conformation of the β1-integrin. To our knowledge, this is the first report demonstrating that any Notch member can regulate inside-out signaling of integrins. We are currently in the process of examining the potential pathways contributing to modulation of β1-integrin affinity by Notch4.
There is much evidence demonstrating that suppression of integrin activation is a physiological mechanism with which to control integrin-dependent cell adhesion and migration (33
). In addition, regulation of integrin activation has been reported to precede differentiation in several cell types. Regulation of β1-integrin activity in neurogenic and myogenic differentiation, two processes that are also modulated by Notch, has been reported (8
). In a baboon model, it has previously been shown that in uninjured saphenous arteries endothelial cells and vascular smooth muscle cells express an epitope characteristic of β1-integrins in a high-affinity state (43
). However, 6 weeks following balloon injury, regenerating endothelial cells did not express this ligand-induced epitope, although there was no decrease in the expression of total β1-integrin (43
). In the same study, activation of β1-integrin with function-activating β1 antibody 8A2 inhibited the migration of endothelial cells in vitro (43
). Together, these findings suggest that activated β1-integrin is required to maintain endothelial cells in a quiescent state, but, to repair arteries and possibly to allow neovascularization, dyshesion by downregulating β1-integrin affinity is required. In fact, activation of β1-integrins on human endothelial cells has been shown to inhibit capillary tube formation in collagen gels in vitro (25
). We report that activation of β1-integrins on endothelial cells, independent of Notch4 activation, inhibits endothelial sprouting in vitro (Fig. ). Furthermore, we demonstrate that β1-integrin activation can inhibit angiogenesis in the chick CAM in vivo (Fig. ). In a previous study using function-blocking antibodies directed against specific α-integrin subunits, a combination of α1-blocking and α2-blocking antibodies was shown to inhibit VEGF-induced angiogenesis in a mouse Matrigel plug assay (66
). These findings suggest that blocking α1β1- and α2β1-integrin function can inhibit VEGF-induced angiogenesis (66
). Although these results may seem contradictory to our data demonstrating that blocking β1-integrin function does not inhibit VEGF-induced angiogenesis in the chick CAM (Fig. ), it is important to note that the effect of function-blocking and -activating antibodies directed against the β1-integrin subunit in the Matrigel plug assay was not reported. Because numerous αβ1-integrin heterodimers are implicated in angiogenesis (4
), blocking the function of only the α1 and α2 subunits may result in a different phenotype from that seen when the function of all β1-integrins is blocked. Alternatively, the different results may reflect intrinsic differences in the experimental models used. Indeed, function-blocking β1-integrin antibody CSAT has been reported to disrupt vascular development and lumen formation when microinjected into quail embryos (18
), whereas the same CSAT antibody does not affect FGF-2- or tumor necrosis factor alpha-induced angiogenesis in the chick CAM (10
Because Notch4 expression is restricted to the endothelium (74
) and because Notch4 is the only Notch receptor expressed in the capillary endothelium (76
), our findings implicate selective activation of Notch4 as a possible method by which to inhibit angiogenesis in pathological contexts. However, because our studies involve a constitutively active, overexpressed form of Notch4 in endothelial cells, the physiological relevance of the data must be interpreted with caution. Further studies using ligands specific for Notch4 will be important to determine whether modulated activation of Notch4 also inhibits angiogenesis. Recent studies suggest that Delta-like4 (Dll4) may be a potential ligand for Notch4, based on similar expression patterns for the two proteins (45
). However, it remains to be seen whether Dll4 can physically interact with and activate Notch4 and induce Notch4 signaling.
Hence the ability of Notch4 to inhibit endothelial sprouting in vitro and angiogenesis in vivo may be related in part to its ability to increase the ligand-binding affinity of β1-integrins, as we have demonstrated in this report. Other potential mechanisms, however, may act in concert with β1-integrin activation to mediate the observed Notch4 effect.