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First published online 4 July 2007
doi: 10.1242/dev.004184
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Review |
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA.
e-mail: tom.gridley{at}jax.org
SUMMARY
Notch signaling is an ancient intercellular signaling mechanism that plays myriad roles during vascular development and physiology in vertebrates. These roles include regulation of artery/vein differentiation in endothelial and vascular smooth muscle cells, regulation of blood vessel sprouting and branching during both normal development and tumor angiogenesis, and the differentiation and physiological responses of vascular smooth muscle cells. Defects in Notch signaling also cause inherited vascular and cardiovascular diseases. In this review, I summarize recent findings and discuss the growing relevance of Notch pathway modulation for therapeutic applications in disease.
Introduction
The Notch signaling pathway is an evolutionarily conserved, intercellular signaling mechanism essential for proper embryonic development in all metazoan organisms in the Animal kingdom. Notch signaling frequently plays a crucial role in precursor cells making binary cell fate decisions. However, Notch signaling also regulates boundary formation between cell populations, cell proliferation and cell death. In addition, perturbations in Notch signaling contribute to the pathogenesis of several inherited human diseases and cancers. In this review, I will highlight the multiple roles that the Notch signaling pathway plays during vascular development and physiology in vertebrates.
Core components of the Notch signaling pathway
Notch family receptors are large single-pass type I transmembrane proteins (Fig. 1). In mammals, four Notch family receptors have been described: NOTCH1 through to NOTCH4. The extracellular domain of Notch family proteins contains up to 36 tandemly repeated copies of an epidermal growth factor (EGF)-like motif. Each Notch family receptor exists at the cell surface as a proteolytically cleaved heterodimer comprising a large ectodomain and a membrane-tethered intracellular domain. Notch receptors interact with single-pass type I transmembrane ligands expressed on neighboring cells. This restricts the Notch pathway to regulating short-range intercellular interactions. In mammals, the Notch ligands are encoded by the Jagged (JAG1 and JAG2) and Delta-like (DLL1, DLL3 and DLL4) gene families.
Upon ligand binding, a signal is transmitted intracellularly by a process
involving the proteolytic cleavage of the receptor and the subsequent nuclear
translocation of the Notch intracellular domain (NICD). The receptor-ligand
interaction induces two additional proteolytic cleavages in the
membrane-tethered fragment of the Notch heterodimer. The final cleavage,
catalyzed by the 
-secretase complex, frees the NICD from the cell
membrane. This cleaved fragment translocates to the nucleus because of the
presence of nuclear localization signals located in the NICD. Once in the
nucleus, the NICD forms a complex with the recombination signal binding
protein for immunoglobulin kappa J region (RBPJ) protein - a sequence-specific
DNA-binding protein. In the absence of the NICD, the RBPJ protein binds to
specific DNA sequences in the regulatory elements of various target genes and
represses transcription of these genes by recruiting histone deacetylases and
other components to form a co-repressor complex. The nuclear translocation of
the NICD displaces the histone deacetylase-co-repressor complex from the RBPJ
protein. The NICD-RBPJ complex recruits other proteins, such as the
mastermind-like 1 (MAML1) protein and histone acetyltransferases, leading to
the transcriptional activation of Notch target genes. Among the most commonly
induced Notch target genes are the basic helix-loop-helix (bHLH)
transcriptional repressors of the hairy and enhancer of split/hairy and
enhancer of split related with YRPW motif (Hes/Hey) family
(Kageyama et al., 2007
).
Further details on the biochemistry of the Notch signaling pathway can be
found in several recent reviews (Bray,
2006; Ehebauer et al.,
2006a; Ehebauer et al.,
2006b; Ilagan and Kopan,
2007; Kageyama et al.,
2007; Le Borgne,
2006).
Artery-vein differentiation
A role for the Notch pathway in regulating vascular development was
initially suggested based on findings from the analysis of several targeted
mouse mutants in Notch pathway components. Mouse mutants for which targeted
mutagenesis and transgenic studies have demonstrated a role in embryonic
vascular development include the receptors Notch1
(Huppert et al., 2000;
Krebs et al., 2000;
Limbourg et al., 2005) and
Notch4 (Carlson et al., 2005;
Krebs et al., 2000;
Uyttendaele et al., 2001); the
ligands Jag1 (Xue et al.,
1999) and Dll4 (Duarte et al.,
2004; Gale et al.,
2004; Krebs et al.,
2004); the Notch transcriptional regulator Rbpj
(Krebs et al., 2004); the E3
ubiquitin ligase Mib1 (Barsi et al.,
2005; Koo et al.,
2005); components of the -secretase complex, such as
nicastrin (Li et al., 2003),
presenilin 1 and presenilin 2 (Herreman et
al., 1999); and the Notch pathway downstream effector bHLH
proteins Hey1 and Hey2 (Fischer et al.,
2004; Kokubo et al.,
2005). Most of these mutants exhibit a similar phenotype
characterized by the absence of angiogenic vascular remodeling in the
extraembryonic yolk sac, placenta and embryo proper
(Fig. 2). However, an analysis
of zebrafish embryos with reduced Notch signaling gave the first clues that a
primary function of the Notch pathway during vascular development was to
regulate the specification of arterial fate in endothelial cells.
It had long been believed that the primary factor that regulates the
differentiation of arteries and veins was blood flow. The endothelial cells
that line arteries experience higher blood pressures, higher rates of
hemodynamic flow and higher oxygen tensions than do the endothelial cells that
line veins. However, as described below, recent work has established that
genetic pre-patterning, largely mediated by the Notch pathway, plays a primary
role in regulating arteriovenous differentiation. This genetically determined
pre-pattern is established prior to the initiation of blood flow, but
endothelial cells at this stage are not yet committed to an arterial or venous
cell fate (Jones et al.,
2006). Indeed, recent work has established that, in zebrafish, a
single hemangioblast, the bipotential precursor of a subset of hematopoietic
and endothelial cells, can give rise to endothelial cell progeny that populate
both arteries and veins (Vogeli et al.,
2006).
|
; Coultas et al.,
2005; Shibuya and
Claesson-Welsh, 2006). The roles and interdependence of the Notch
and VEGFA pathways in regulating the formation of the large axial blood
vessels of the trunk - the dorsal aorta and the posterior cardinal vein - was
studied first in zebrafish (Lawson et al.,
2001; Lawson et al.,
2002). Notch signaling-deficient embryos exhibit a poorly formed
dorsal aorta and posterior cardinal vein with accompanying arteriovenous
malformations (the fusion of arteries and veins without an intervening
capillary bed). These embryos also exhibited a loss of expression of arterial
markers such as ephrin B2 from arterial vessels with an accompanying expansion
of venous markers into normally arterial domains. Embryos in which Notch
signaling had been ectopically activated exhibited the reverse phenotype:
suppression of vein-specific markers with ectopic expression of arterial
markers in venous vessels (Lawson et al.,
2001). A similar phenotype was observed in embryos mutant for
certain Notch target genes, such as the bHLH transcriptional repressor
hey2 (also referred to in zebrafish as the gridlock gene)
(Zhong et al., 2001;
Zhong et al., 2000).
This analysis of the formation of the major trunk vessels in the zebrafish
embryo revealed a signaling cascade that is responsible for determining
arterial and venous cell fates in these vessels
(Lawson et al., 2002)
(Fig. 3). A reduction in
vegfa activity results in a loss of arterial marker expression from
the dorsal aorta and in the ectopic arterial expression of vein markers.
Conversely, the injection of vegfa mRNA induces ectopic expression of
the arterial marker ephrin B2 in the posterior cardinal vein.
vegfa expression is regulated by the expression of the secreted
morphogen sonic hedgehog a (shha) along the axial midline.
Similar to what is observed in vegfa-deficient embryos, shha
mutant zebrafish embryos also exhibit a loss of arterial differentiation,
whereas injection of shha mRNA causes the ectopic expression of
arterial markers. shha acts upstream of vegfa, because the
injection of vegfa mRNA into shha mutant embryos restores
normal arterial differentiation. This work also demonstrated that, in this
setting, the Notch pathway acts downstream of the Vegfa pathway. Whereas
injection of vegfa mRNA into Notch signaling-deficient zebrafish
embryos could not rescue arterial marker gene expression, the expression of an
activated notch1a transgene in vegfa-deficient embryos could
rescue the expression of arterial markers
(Lawson et al., 2002).
Studies in mammalian cell culture have also placed the Notch pathway
downstream of the Vegfa pathway. VEGFA administration induces NOTCH1
and DLL4 expression in human arterial endothelial cells, but not in
venous endothelial cells (Liu et al.,
2003). Targeted-mutagenesis studies in mice have also demonstrated
that Vegfa is essential for vascular development. Mouse embryos heterozygous
for a Vegfa targeted mutation exhibit lethal haploinsufficiency
(Carmeliet et al., 1996;
Ferrara et al., 1996). Blood
vessels, despite forming in these embryos, are severely constricted or
atretic. It is not known whether artery-vein differentiation is compromised in
Vegfa+/- mouse embryos. However, other gain-of-function
transgenic experiments have demonstrated a role of Vegfa in
regulating arterial endothelial cell differentiation in mice. Alternative
splicing of the mouse Vegfa gene results in the production of several
different protein isoforms (Vegfa 120, Vegfa 164 and Vegfa 188). Genetically
engineered mice that express only the Vegfa 164 isoform display normal retinal
vascular development. However, mice that express only Vegfa 120 exhibit severe
defects in retinal vascular outgrowth, whereas mice expressing only Vegfa 188
exhibit impaired retinal arterial development, but normal venous development
(Stalmans et al., 2002). Vegfa
164 overexpression in cardiac muscle increased the number of ephrin B2
(Efnb2)-positive capillaries in the mouse heart while reducing the number of
ephrin receptor B4 (EphB4)-positive venules
(Visconti et al., 2002). Vegfa
could induce ephrin B2 gene expression in mouse primary embryonic endothelial
cells, and Vegfa derived from sensory neurons, motor neurons and Schwann cells
is required for arterial differentiation of small-diameter nerve-associated
vessels in mice (Mukouyama et al.,
2005; Mukouyama et al.,
2002).
|
;
Gale et al., 2004;
Krebs et al., 2004). However,
some Dll4+/- mice are viable on an outbred background,
permitting the examination of Dll4-/- embryos. The
phenotype of Dll4-/- homozygotes was similar, although
more severe, than that of Dll4+/- heterozygous embryos
(Duarte et al., 2004;
Gale et al., 2004). Similar to
that which is observed in Notch signaling-deficient zebrafish embryos, both
Dll4-deficient embryos and other types of Notch signaling-deficient
mouse embryos, such as Rbpj mutant and Hey1; Hey2
double-mutant embryos, do not express arterial markers
(Duarte et al., 2004;
Fischer et al., 2004;
Gale et al., 2004;
Kokubo et al., 2005;
Krebs et al., 2004). In
support of a direct role for Notch signaling in regulating the expression of
important arterially expressed genes, Dll4-mediated Notch signaling induces
ephrin B2 gene expression in cultured endothelial cells
(Iso et al., 2006), and the
ephrin B2 gene has recently been shown to be a direct Notch target
(Grego-Bessa et al.,
2007).
Little is known about the transcriptional regulation of genes that exhibit
arterially restricted expression in early embryos. The forkhead (Fox; also
known as winged helix) proteins are a large family of evolutionarily conserved
transcription factors (Kaestner et al.,
2000). Mouse embryos with compound mutations of the Foxc1
and Foxc2 genes, two related Fox family transcription factors,
display defects in vascular remodeling in the yolk sac and embryo
(Kume et al., 2001),
accompanied by reduced or absent expression of arterial markers and the
occurrence of arteriovenous malformations
(Seo et al., 2006). This
failure of arterial specification is likely to be due to disrupted regulation
of Dll4 transcription. The Foxc1 and Foxc2 proteins directly activate
Dll4 transcription through a Foxc-binding element in the upstream
region of the Dll4 gene. These results demonstrate that the Foxc
proteins are key transcriptional regulators that act upstream of the Notch
pathway during arteriovenous differentiation
(Seo et al., 2006). However,
it is not known whether Foxc1 and Foxc2 expression is
downstream, or independent of, Vegfa signaling.
As mentioned previously, Notch signaling-deficient zebrafish embryos form
arteriovenous malformations. Arteries normally connect to veins only through
an intervening capillary bed. An aberrant direct communication between an
artery and vein is termed an arteriovenous malformation. One mechanism that
might explain the formation of arteriovenous malformations is the failure to
establish or maintain distinct arterial and venous vascular beds. In mouse
embryos, injection of ink into the heart is an effective way to visualize the
presence of arteriovenous malformations
(Sorensen et al., 2003). In
Notch signaling-deficient mouse embryos (e.g. Dll4+/- or
Notch1-/- embryos) arteriovenous malformations that are
also detectable by histological analysis
(Duarte et al., 2004;
Gale et al., 2004;
Krebs et al., 2004) form
(Fig. 4)
(Krebs et al., 2004).
Interestingly, the inducible expression of an activated Notch4
transgene in adult mice results in vessel arterialization, such as the
induction of venous expression of the ephrin B2 gene, and causes arteriovenous
malformations in several organs, including liver, uterus and skin
(Carlson et al., 2005).
Surprisingly, these malformations are reversible if activated Notch4
transgene expression is repressed. These studies demonstrate that the ability
of Notch signaling to arterialize blood vessels is not confined to the
embryonic period. Although gain-of-function in vivo assays using the
expression of an activated Notch4 transgene can cause mutant vascular
phenotypes (Carlson et al.,
2005; Uyttendaele et al.,
2001), it should be noted that no obvious phenotype is observed in
Notch4-/- mice (Krebs
et al., 2000).
In addition to regulating arterial specification of endothelial cells,
Notch signaling also regulates arterial specification of vascular smooth
muscle cells. The Notch3 gene is expressed in vascular smooth muscle
cells of arteries, but not in those of veins. Marked arterial defects occur in
Notch3-/- mice, including enlarged arteries with a thinner
vascular smooth muscle cell coat than is found in wild-type arteries
(Domenga et al., 2004). These
defects arise postnatally, because arterial vessels fail to mature.
Morphologically, arterial vascular smooth muscle cells of
Notch3-/- mice resemble those surrounding veins in
wild-type mice. Only a few markers are known to be expressed predominantly in
arterial vascular smooth muscle cells and not in venous ones. These include
smoothelin (van der Loop et al.,
1997) and a transgenic line expressing the ß-galactosidase
protein from arterial-specific regulatory elements of the SM22
promoter
(Moessler et al., 1996). The
expression of both of these markers is markedly downregulated in arteries of
Notch3-/- mice (Fig.
5). Combined with the morphological data, this indicates that
vascular smooth muscle cells that surround arteries in
Notch3-/- mice have acquired a venous fate. Notably, in
arteries of Notch3-/- mice, which do not express arterial
markers for vascular smooth muscle cells, normal expression of several
endothelial cell arterial markers occurs, including that of ephrin B2,
connexin 40 (also known as Gja5 - Mouse Genome Informatics), Hes1, Hey1, Hey2
and Heyl (Domenga et al.,
2004). These results demonstrate that the arterial identity of
endothelial cells, and of the vascular smooth muscle cells surrounding them,
is specified independently.
Endothelial tip cell differentiation
During angiogenesis, new capillaries sprout from existing blood vessels.
Tip cells are specialized endothelial cells situated at the tips of vascular
sprouts that extend filopodia in response to cues within the local
extracellular environment, guiding the growth of these sprouts along Vegfa
gradients (Gerhardt et al.,
2003; Gerhardt et al.,
2004). Recent work has identified a primary role for the Notch
pathway in regulating the formation and function of these endothelial tip
cells, a role that was first described several years ago
(Sainson et al., 2005). In an
in vitro angiogenesis culture system using human umbilical vein endothelial
cells (HUVECs), Notch signaling inhibits branching at the tip of developing
angiogenic sprouts. The suppression of Notch signaling led to tip cell
division, with both daughter cells being specified as tip cells. This
subsequently led to increased branching as a result of vessel bifurcation.
|
;
Lobov et al., 2007;
Ridgway et al., 2006;
Suchting et al., 2007); the
zebrafish embryo (Leslie et al.,
2007; Siekmann and Lawson,
2007); and xenograft tumor models
(Noguera-Troise et al., 2006;
Ridgway et al., 2006;
Scehnet et al., 2007). A
finding common to all of these studies is the increased sprouting and
branching of blood vessels when Notch signaling is inhibited. The Notch
pathway regulates sprouting and branching behaviors in these vessels by
influencing the differentiation, migration and proliferation of vascular tip
cells; reduced Notch signaling leads to increases in tip cell numbers,
filopodia extension and vessel branching. Suppression of tip cell formation
and angiogenic sprouting by Notch signaling is downstream of the VEGFA signal,
because pharmacological or genetic manipulations that block VEGFA function
reduce both DLL4 expression and blood vessel sprouting.
Several of these recent studies have assessed the effects of modulating
Notch signaling on the differentiation of the developing retinal vasculature
in mice (Hellstrom et al.,
2007; Lobov et al.,
2007; Ridgway et al.,
2006; Suchting et al.,
2007). Compared with other organs, the mouse retina possesses
several distinct advantages for the analysis of developmental angiogenesis
(Dorrell and Friedlander,
2006; Gariano and Gardner,
2005; Uemura et al.,
2006). The mouse retinal vascular system develops postnatally in a
highly reproducible spatial and temporal pattern. The retinal vascular system
emerges first in the region of the optic nerve head, and then grows radially
towards the periphery. The primitive vascular plexus that forms initially is
then remodeled into large and small arterial and venous vessels. During these
stages, the retinal vasculature is accessible both for observation and for the
experimental administration of exogenous agents.
The Dll4 gene is highly expressed in the developing retinal
vasculature, and reduced Dll4/Notch signaling leads to striking defects in the
early postnatal retinal vasculature. The observed defects are concordant
whether Dll4/Notch signaling is reduced genetically, by assessing
Dll4+/- heterozygous mice
(Hellstrom et al., 2007;
Lobov et al., 2007;
Suchting et al., 2007) or mice
with temporally-regulated Notch1 deletion in the retinal vasculature
(Hellstrom et al., 2007), or
by administering anti-Dll4 blocking reagents
(Lobov et al., 2007;
Ridgway et al., 2006) or
-secretase inhibitors (Hellstrom et
al., 2007; Suchting et al.,
2007). The retinal vasculature in these mice displays severe
patterning defects (Fig. 6).
The vascular plexus has an increased capillary density and diameter, with
increased filopodial extensions both at the growing vascular front, and in the
interior of the plexus. Furthermore, portions of the vascular plexus fuse to
form syncytial sinuses. Markers specific for tip cells, such as platelet
derived growth factor receptor beta (Pdgfrb) and unc-5 homolog B
(Unc5b), are also upregulated in mice with reduced Dll4/Notch
signaling. These data indicate that Dll4/Notch signaling restricts the
acquisition of an endothelial tip cell fate in angiogenic sprouts.
|
|
;
Siekmann and Lawson, 2007).
The optical clarity of these embryos and the availability of several
transgenic lines that express fluorescent proteins in endothelial cells makes
this animal model an ideal system for high-resolution fluorescent microscopy,
including live time-lapse confocal microscopy. Mosaic analysis has revealed
that transplanted cells that lack rbpj function do not contribute to
the dorsal aorta, but become located preferentially in the posterior cardinal
vein or the most dorsal position of the segmental arteries. Conversely,
transplanted cells that express activated Notch1 become located preferentially
in the dorsal aorta or the base of developing sprouts
(Siekmann and Lawson, 2007).
These results indicate that Notch signaling is required cell-autonomously to
specify endothelial cell fate in segmental artery sprouts. The use of
time-lapse confocal microscopy on living embryos revealed that, in both
dll4 morphant and rbpj morphant embryos, segmental artery
sprouts contain more cells than in controls. These additional cells are
incorporated via both the increased migration of endothelial cells into the
initial sprout, and by the proliferation of normally quiescent stalk cells.
Interestingly, vascular defects in rbpj morphant embryos are more
severe than those in dll4 morphant embryos, suggesting that
additional Notch ligands play important roles during early vascular
development (Leslie et al.,
2007; Siekmann and Lawson,
2007). Blocking Vegfa signaling with a small molecule inhibitor
prevents both normal endothelial sprouting and the ectopic sprouting observed
in dll4 morphant embryos (Leslie
et al., 2007). In addition, the reduction of Vegf receptor 3 (also
known as Flt4 - Zebrafish Information Network) levels in rbpj
morphant embryos partially rescues the rbpj-knockdown phenotype,
suggesting that Notch activation might normally repress Vegf receptor 3 and
thus limit angiogenic cell behavior in developing segmental artery sprouts
(Siekmann and Lawson, 2007).
Taken together, the studies in both the mouse retina and the zebrafish embryo
indicate that Notch signaling acts as a negative regulator of Vegfa-induced
angiogenesis, and is essential for proper vascular morphogenesis. Tumor angiogenesis
The maintenance, growth and metastasis of solid tumors require the
recruitment of host blood vessels into the tumor. Many solid tumors express
VEGFA, and therapies that use anti-Vegfa antibodies or other blocking reagents
effectively inhibit solid tumor growth in preclinical rodent models
(Ferrara and Kerbel, 2005;
Jain et al., 2006). Given the
prominent role of the Notch pathway in regulating vascular development,
protein components of the Notch pathway might provide novel drug targets
during tumor angiogenesis. Dll4 is expressed at high levels in tumor
vasculature (Gale et al.,
2004; Hainaud et al.,
2006; Mailhos et al.,
2001; Patel et al.,
2005), and several recent studies have identified the Dll4 protein
as just such a therapeutic target
(Noguera-Troise et al., 2006;
Ridgway et al., 2006;
Scehnet et al., 2007). The
systemic administration of neutralizing anti-Dll4 antibodies
(Noguera-Troise et al., 2006;
Ridgway et al., 2006), and the
systemic (Noguera-Troise et al.,
2006) or localized (Scehnet et
al., 2007) administration of recombinant forms of the Dll4 protein
that have been modified to block Dll4/Notch signaling, inhibit the growth of
several different solid tumors in mice. Similar to the findings in zebrafish
embryos and mouse retinas, anti-Dll4 treatment increases blood vessel
sprouting and branching, and leads to a marked increase in tumor blood vessel
density in the treated tumors. Paradoxically, tumor growth was inhibited in
these mice despite the increased blood vessel density. Analysis of the
vascular network in the anti-Dll4-treated tumors by perfusion assays with
fluorescent lectins or by assessing hypoxic regions in these tumors revealed
that the newly induced vessels function inefficiently. Many of these vessels
are not connected to the vascular network in the tumors, leading to poor
perfusion, increased hypoxia and an overall inhibition of tumor growth.
Importantly, anti-Dll4 therapies are effective against tumors that were
resistant to anti-VEGFA treatments, and could provide synergistic effects
against certain tumors when combined with anti-Vegfa therapies
(Noguera-Troise et al., 2006;
Ridgway et al., 2006).
|
; Jain et al.,
2006). Similar issues might arise as anti-DLL4 treatments progress
from preclinical models into human clinical trials. However, these promising
studies present a novel therapeutic approach for cancer treatment,
particularly for tumors that are unresponsive to anti-VEGFA therapies. Vascular smooth muscle cell differentiation and physiology
It is clear that Notch signaling plays an important role in the
differentiation, physiology and function of vascular smooth muscle cells.
However, contradictory results suggest that its role might be context-, time-
or cell line-dependent. Several groups have described a role for Notch
signaling in repressing smooth muscle cell differentiation during in vitro
cell culture, and that this repressive effect is likely to be mediated via the
induction of the HEY2 protein (Doi et al.,
2005; Morrow et al.,
2005a; Proweller et al.,
2005). However, more-recent studies have indicated that Notch
signaling induces smooth muscle cell differentiation
(Doi et al., 2006;
High et al., 2007);
JAG1-mediated Notch signaling is reported to promote smooth muscle cell
differentiation both in human aortic smooth muscle cells and in a murine
embryonic fibroblast cell line (Doi et
al., 2006). Both smooth muscle myosin heavy chain
(Doi et al., 2006) and smooth
muscle -actin (Noseda et al.,
2006) were demonstrated to be direct Notch target genes.
Furthermore, in vivo studies in which Notch signaling was inactivated
specifically in mouse neural crest cells reveals that Notch signaling plays an
essential role in the differentiation of cardiac neural crest cells into
smooth muscle cells (High et al.,
2007).
Several studies have characterized the expression of Notch pathway genes
during the response to vascular injury
(Campos et al., 2002;
Doi et al., 2005;
Lindner et al., 2001;
Wang et al., 2002). The
expression of several Notch pathway components, including Notch1, Notch3,
Jag1, Jag2, Hey1 and Hey2, is modulated after experimentally induced vascular
injury. The expression of the genes encoding these proteins is downregulated
within the first 2 days following vascular injury, but is upregulated in
comparison to uninjured contralateral control vessels at 7-14 days after
injury. In support of a functional role for the modulation of Notch pathway
components during the response to vascular injury, formation of the neointima
(a thickened layer of vascular smooth muscle cells) after vascular injury was
significantly decreased in Hey2-/- mice
(Sakata et al., 2004). The
culture of primary aortic vascular smooth muscle cells from
Hey2-/- mice has revealed that these mutant cells
proliferate at a reduced rate compared with wild-type cells. The
overexpression of Hey1 (Wang et
al., 2003) or Hey2
(Havrda et al., 2006) in
vascular smooth muscle cells led to increased vascular smooth muscle cell
proliferation associated with reduced levels of the cyclin-dependent kinase
inhibitors p21waf1/cip1 (Cdkn1a)
(Wang et al., 2003) or
p27kip1 (Cdkn1b)
(Havrda et al., 2006). The
Hey2 protein directly interacts with the p27kip1 promoter
to repress transcription (Havrda et al.,
2006).
Mechanical forces are one of several factors implicated in regulating
vascular smooth muscle cell differentiation and physiology. Adult vascular
smooth muscle cells are not terminally differentiated and can exhibit
substantial plasticity in their phenotype in response to local environmental
changes. The exposure of primary human or rat vascular smooth muscle cells to
cyclic mechanical strain during in vitro culture causes a significant
reduction in NOTCH1 and NOTCH3 receptor expression, concomitant with an
increase in the expression of vascular smooth muscle cell differentiation
markers (Morrow et al., 2005a;
Morrow et al., 2005b).
Vascular smooth muscle cells that are exposed to mechanical strain also
exhibit reduced proliferation and increased apoptosis. Overexpression of the
NOTCH1 or NOTCH3 intracellular domains in vascular smooth muscle cells exposed
to such mechanical strain restored the percentages of proliferative or
apoptotic cells to the levels observed in unstrained cells. These results
indicate that cyclic mechanical strain inhibits vascular smooth muscle cell
growth while increasing apoptosis, and that these effects are mediated, at
least in part, via the modulation of Notch signaling.
Vascular smooth muscle cell physiology: CADASIL
The importance of Notch signaling in vascular development is highlighted by
the finding that mutations in several Notch pathway components cause inherited
vascular or cardiovascular diseases. The vascular defects associated with two
of these inherited diseases, Alagille syndrome and inherited bicuspid aortic
valve, primarily affect the development and function of the heart
(Box 1). A third disease, which
is caused by mutations in the NOTCH3 gene
(Joutel et al., 1996), is an
inherited degenerative vascular disease that affects vascular smooth muscle
cells. Cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy, termed CADASIL, is the most common genetic form of stroke
and vascular dementia (Kalaria et al.,
2004). Affected individuals exhibit a variety of symptoms,
including migraines, mood disorders, recurrent subcortical ischemic strokes,
progressive cognitive decline, dementia and premature death. CADASIL is
characterized by the progressive degeneration of vascular smooth muscle cells
and the accumulation of granular osmiophilic material (GOM) within the smooth
muscle cell basement membrane (Kalaria et
al., 2004). GOM accumulation in vascular smooth muscle cells is
one of the most distinguishing features of CADASIL.
All NOTCH3 mutations associated with CADASIL result in a gain or
loss of a cysteine residue in one of the 34 EGF-like repeats in the
extracellular domain of the NOTCH3 receptor. The characteristic nature of
these mutations, in addition to the absence of any examples in CADASIL
patients of mutations or deletions of the NOTCH3 gene that are
obviously inactivating, strongly suggests that mutations that cause CADASIL
are not NOTCH3-null alleles. In CADASIL patients, the ectodomain of
the NOTCH3 protein accumulates in the cerebral microvasculature at the
cytoplasmic membrane of vascular smooth muscle cells
(Joutel et al., 2000),
although it is controversial whether the NOTCH3 ectodomain constitutes part or
all of the GOM deposits (Ishiko et al.,
2006; Joutel et al.,
2000).
Two different mouse models that express NOTCH3 proteins containing
mutations found in CADASIL patients have been developed. In one model, an
Arg142Cys knock-in mutation was introduced into the endogenous mouse
Notch3 gene (Lundkvist et al.,
2005). These mice do not exhibit any CADASIL-like morphological or
behavioral phenotypes, even when homozygous for this mutation. The second
model more successfully recapitulates the early, preclinical phase of CADASIL.
To create this model, transgenic mice were generated that express a human
NOTCH3 cDNA that contains a different CADASIL mutation, the Arg90Cys
mutation, in vascular smooth muscle cells
(Ruchoux et al., 2003). An
age-dependent accumulation of the NOTCH3 ectodomain and of GOM deposits in
vascular smooth muscle cells of both cerebral and peripheral arterioles is
observed in these mice. However, despite GOM accumulation, no evidence of
damage to the brain parenchyma is seen. Physiological studies of these NOTCH3
Arg90Cys transgenic mice revealed an impaired cerebral vasoreactivity that
suggests either decreased relaxation or increased resistance of cerebral blood
vessels (Lacombe et al.,
2005). Isolated caudal arteries from the tails of these mice
exhibit increased pressure-induced contraction and decreased flow-induced
dilation (Dubroca et al.,
2005). Transgenic mice that express either the wild-type human
NOTCH3 protein or the human NOTCH3 Arg90Cys mutation were equally effective in
rescuing the arterial defects of Notch3-/- mice.
Furthermore, the expression of the mutant NOTCH3 Arg90Cys protein correctly
regulates the in vivo expression of a Notch signaling reporter
(Monet et al., 2007). These
data suggest that novel pathogenic roles for CADASIL mutant proteins, rather
than compromised NOTCH3 signaling activity, underlie the etiology of CADASIL.
Further analysis and development of the NOTCH3 Arg90Cys mouse model should
lead to valuable insights into the onset and progression of CADASIL,
particularly during its early, preclinical stages.
| Box 1. Notch signaling and inherited cardiovascular disease
Alagille syndrome is an autosomal dominant disorder characterized by
developmental abnormalities of the liver, heart, eye, skeleton and, at lower
penetrance, several other organs (Gridley,
2003
Bicuspid aortic valve affects 1-2% of the population, making it the most
common congenital cardiac malformation
(Braverman et al., 2005
|
Conclusion
As can be seen by the recent flurry of publications that focus on the role
of Notch signaling in regulating endothelial tip cell formation and the
resulting implications for tumor angiogenesis
(Hellstrom et al., 2007;
Leslie et al., 2007;
Lobov et al., 2007;
Noguera-Troise et al., 2006;
Ridgway et al., 2006;
Scehnet et al., 2007;
Siekmann and Lawson, 2007;
Suchting et al., 2007), we
still have much to learn about the roles that the Notch pathway plays in the
vasculature. How are the different roles that the Notch pathway plays in, for
example, arteriovenous patterning, tip cell differentiation, and vessel wall
formation, integrated during vascular development and physiology? Are these
roles related mechanistically, or do they represent different aspects of Notch
pathway function? What is the contribution of Notch signaling during normal
vascular physiology in adults, and how do perturbations in Notch signaling
contribute to vascular pathology and disease?
Areas in which there will certainly be significant advances in upcoming years include the development of anti-DLL4 therapies (and possibly other types of `anti-Notch' therapies) that target tumor angiogenesis as well as other vascular diseases characterized by pathological angiogenesis, such as neovascular age-related macular degeneration and diabetic retinopathy. We are also likely to see the development and characterization of better animal models for CADASIL. Because CADASIL causes the most common form of inherited stroke and vascular dementia in humans, animal models that better recapitulate the pathological and behavioral abnormalities exhibited by CADASIL patients will permit the development and evaluation of therapeutic treatment regimens both for CADASIL and for sporadic cases of vascular dementia. Significant advances in our understanding of the mechanisms for cross-talk between the Notch pathway and other signaling pathways, such as the TGFß, Wnt, ephrin/Eph receptor and PI3K/Akt pathways, and in the developmental and physiological decisions in which such cross-talk is operative, are also likely to occur in the near future. The answers to these and other questions will keep Notch pathway researchers occupied for many years to come.
ACKNOWLEDGMENTS
I apologize to colleagues whose work could not be cited directly due to space constraints. I thank Anne Joutel, Anne Eichmann and Luke Krebs for images for some of the figures. Work in my laboratory on the Notch pathway is supported by the National Institutes of Health.
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