ARTICLE
Auteur(s) : Yasufumi Sato
Department of Vascular Biology, Institute
of Development, Aging and Cancer, Tohoku University,
Sendai, Japan
accepté le 12 Juin 2009
The vascular system, a hierarchical network of arteries,
capillaries and veins, is one of the most quiescent organs in the
body, but it has the capacity to form neo-vessels under certain
conditions. Angiogenesis or neovascularization, i.e., the formation
of neovessels, is a fundamental process that occurs in the vascular
system, and it occurs under both physiological and pathological
conditions. Angiogenesis can be classified into sprouting
angiogenesis and intussusceptions. Although the process resulting
in intussusceptions has been poorly investigated, that of sprouting
angiogenesis is better characterized at present.
The vascular system is primarily composed of luminal endothelial
cells (ECs) and surrounding mural cells (smooth muscle cells or
pericytes). The presence of mural cells causes blood vessels to
become mature and stabilized. The initial step of sprouting
angiogenesis is the detachment of mural cells for vascular
destabilization. Thereafter, specialized ECs, so-called tip cells,
start to migrate by extending numerous filopodia, whereas following
ECs, so-called stalk cells, proliferate, cause elongation of
sprouts, and form immature, tube-like structures. Finally,
redistributed mural cells attach to newly formed vessels for
vascular restabilization. By this final process, ECs stop their
proliferation, thus terminating angiogenesis.
Angiogenesis is thought to be regulated by the local balance
between stimulators and inhibitors of this process. A number
of endogenous angiogenesis stimulators and inhibitors have been
found in the body. Angiogenesis stimulators include certain growth
factors and cytokines, whereas angiogenesis inhibitors are varied
and include hormones, chemokines, proteolytic fragments of various
proteins, proteins accumulated in the extracellular matrix, and so
forth [1]. The origin of these angiogenesis inhibitors is mostly
extrinsic to the vasculature. Recently however, ECs have been found
to produce angiogenesis inhibitors by themselves, including
delta-like 4 and vasohibin 1 (see below). Such intrinsic
factors may regulate angiogenesis in an autoregulatory or
negative-feedback fashion.
Delta-like 4 (Dll4)
The Notch-signaling system is evolutionarily conserved from
Drosophila to humans, regulating cell fate specification, growth,
differentiation, and patterning of neighboring cells through
lateral inhibition. The Notch-signaling system in mammals consists
of four type I transmembrane receptors (Notch1, Notch2, Notch3, and
Notch4) and five type I transmembrane ligands (Jagged1, Jagged2,
Dll1, Dll3, and Dll4) collectively referred to as the DSL
(Delta/Serrate/Lag-2) family. The Notch receptor consists of an
extracellular domain and an intracellular domain. The Notch
extracellular domain (NECD) is composed of epidermal growth factor
(EGF)-like repeats, followed by Lin12-Notch (LN) repeats. These
EGF-like repeats contain the ligand-binding sites, whereas the LN
repeats are involved in preventing ligand-independent signaling. On
the other hand, the Notch intracellular domain (NIC) contains
recombination signal binding protein for the immunoglobulin kappa J
region (RBPJκ)-associated molecular region in the juxtamembrane
region, followed by ankyrin repeats, a putative transactivating
domain, and a C-terminal PEST motif. PEST is defined by a cluster
of proline (P), glutamic acid (E), serine (S) and threonine (T)
residues. Upon Notch receptor-ligand binding at the cell surface, a
series of sequential cleavages of the Notch receptor occurs. The
final cleavage is mediated by the γ-secretase complex, which
results in the release of the NICD, which is then translocated to
the nucleus, where it interacts with members of the CSL (CBF-1,
Suppressor of Hairless, Lag-1) family of transcription factors. The
best-characterized CSL family members of Notch targets are the
Hairy and enhancer-of-split (HES), and Hairy and
enhancer-of-split-related (HEY, HESR, HRT, or CHF) gene families.
These basic, helix-loop-helix (bHLH) proteins act mostly as
transcriptional repressors, either by direct binding to an E-box
and N-box for the recruitment of corepressors such as groucho (TLE
in mammals) or by mechanisms independent of direct DNA binding.
Thus, the interaction of the NICD with CSL family members results
in the derepression/activation of CSL targets [2].
Multiple Notch receptors and ligands are expressed in the
vascular system during both embryonic development and postnatal
remodeling. Among them, Notch2, Notch4, Jagged1, and
Jagged2 expression are restricted mainly to arterial
endothelium, whereas Notch1 and Dll4 are expressed in
both capillary and arterial endothelium [3, 4]. Consistent with
their restricted expression patterns, the Notch-mediated signaling
has been shown to play a critical role in arterial specification.
This activity was initially highlighted in studies on zebrafish.
The blockade of the Notch-mediated signaling in zebrafish embryos
resulted in the loss of arterial markers accompanied by ectopic
expansion of venous markers into arteries [5, 6]. In contrast, the
activation of Notch-mediated signaling exhibited the opposite
effects, suppressing expression of venous markers and promoting
ectopic expansion of arterial markers into veins [5, 6]. Similar
results were also obtained in mice by targeted disruption of
Notch1/Notch4, Rbpsuh encoding RBP-Jκ protein, Hey1/Hey2 or Dll4
[7-11]. Thus, consistent with its restricted expression pattern in
the vasculature, the Notch signaling system plays a critical role
in arterial specification.
Dll4 gene-targeted mice showed an increased number of vessel
branches and vascular sprouts associated with the leading edge of
certain growing vascular beds, such as in the yolk sac [11]. This
phenotype of the Dll4-knockout mice resembled that of the
Notch1-knockout mice [7, 11]. These observations indicate that
Dll4-Notch1-mediated signaling is involved not only in arterial
specification, but also in angiogenesis. Importantly, heterozygous
Dll4-knockout mice show embryonic lethality. Selective disruption
of Notch1 in the endothelium results in embryonic lethality at a
similar time in the development [12], indicating that embryonic
lethality of heterozygous Dll4-knockout mice is closely related to
loss of Notch1 in the endothelium. The precise role of
Dll4-mediated signaling in angiogenesis was characterized further
by studies using zebrafish or newborn mouse retina. These studies
have demonstrate that the expression of Dll4 and
Notch1 are detected mainly in tip cells and stalk cells
respectively, and that Dll4 and Notch1 contribute to the
regulation of tip cells versus stalk cells during sprouting
angiogenesis [13-15]. Notably, the expression of Dll4 and
Notch1 are induced by VEGF [16, 17]. Dll4-mediated signaling
then limits the number of sprouts via Notch1 [14, 18, 19], which
inhibition is attributable to the reduced expression of VEGFR2,
neuropilin-1, and CXCR4 as a negative-feedback regulator [17,
20] (figure 1).
In contrast, the Dll4-Notch1 signal induced Notch-regulated
ankyrin repeat protein (Nrarp) in stalk cells and promoted Wnt
signaling through interactions with lymphoid enhancer factor
1 (Lef1). This Lef1-dependent Wnt signaling in stalk cells is
further involved in the stabilization of newly formed vessels
[21].
The inhibitory role of Dll4 in angiogenesis has also been
documented in tumors. It has been revealed that Dll4 is
up-regulated in the tumor vasculature [22, 23]. When this
Dll4-mediated signaling was blocked, the tumors developed numerous
microvessels. Interestingly, these vessels were non-functioning and
devoid of blood flow, and this unrestrained angiogenesis
paradoxically decreased tumor growth even in certain tumors
resistant to anti-VEGF therapies [24-26]. Alternatively, as
Dll4 is defined as a negative regulator of angiogenesis,
activation of Dll4-mediated signaling can inhibit tumor
angiogenesis and tumor growth in distinct tumor models [27]. So
far, the growth of carcinomas, gliomas, and melanomas has been
reported to have been inhibited by the Dll4/Notch blockade, whereas
that of lymphomas, plasmacytomas, and myelomonocytic tumors has
been reported to be inhibited by Dll4/Notch activation [24, 25,
27]. Future studies will be required to identify the determinants
of responsiveness to Dll4/Notch blockade or activation in various
tumors.
Vasohibin 1 (VASH1)
We hypothesized that ECs might produce novel or uncharacterized
regulators of angiogenesis. To test our hypothesis, we performed
DNA microarray analysis to examine VEGF-inducible genes in ECs
[28]. Among a number of VEGF-inducible genes, we focused our
attention on genes whose functions were previously undefined. We
then performed a functional assay, isolating a protein that showed
antiangiogenic activity, and named it vasohibin (VASH) [29].
Through the subsequent DNA sequence search of genomic databases, we
found one gene homologous to VASH and named it VASH2 [30], and thus
the prototype VASH is now called VASH1. The gene for human VASH is
located on chromosome 14q24.3, and consists of eight exons and
seven introns, which encodes a protein of 365 amino acid
residues. Mouse VASH1 is more than 90% identical to its human
counterpart in amino acid sequence, indicating that VASH1 is
highly conserved at least between humans and mice [29].
A cluster of basic amino acids is present in the C-terminus
region of VASH1 protein, but neither a classical secretion
signal sequence nor any other functional motifs are found in its
amino acid sequence. The lack of a classical signal sequence
suggests that VASH1 is an unconventional secretory protein
[29]. One minor alternative splicing form of VASH1 lacking
exons 5 to 8 is present in humans [30-32]. In addition,
there are multiple different molecular forms that are processed
post-translationally [33].
Immunohistological analysis has revealed that VASH1 is
shown in ECs in the developing embryo and placenta, but is
down-regulated in the postnatal period, and detected in ECs
preferentially at the site of angiogenesis [29, 30, 34]. We further
defined the spacio-temporal expression pattern and function of
VASH1 during angiogenesis. Our analysis, using the mouse
subcutaneous angiogenesis model, has revealed that VASH1 is
expressed not in ECs at the sprouting front (tip and stalk cells),
but in newly formed blood vessels behind the sprouting front where
angiogenesis ceases (termination zone) [35]. Thus, although
Dll4 and VASH1 are expressed in ECs during angiogenesis,
their expression patterns are totally distinctive. We further
demonstrated, in a subcutaneous angiogenesis model, that
VASH1 (-/-) mice contained immature microvessels in the area
where angiogenesis should be terminated [35]. These results
indicate that the central function of endogenous VASH1 is to
terminate angiogenesis (figure 2). Importantly,
newly formed immature microvessels in VASH1 (-/-) mice
function with blood flow [35].
We investigated the expression of VASH1 under various
conditions accompanying pathological angiogenesis. The presence of
VASH1 in ECs was evident in cancers, adventitia of
atherosclerotic lesion, age-dependent macular degeneration (AMD),
and diabetic retinopathy [36-41]. As cancers contain complex
lesions where angiogenesis continues asynchronously and sprouting
occurs randomly, it is difficult to dissect the expression profile
of VASH1. Nevertheless, we showed that VASH1 was prevalent in
tumor vessels of non-small cell lung cancers when they were
associated with mural cells [41]. This observation suggests that
the spacio-temporal expression pattern of VASH1 is maintained
even in tumor angiogenesis. Indeed, tumors inoculated into
VASH1 (-/-) mice contained numerous immature vessels, and this
resulted in increased growth of tumor [41]. In the case of AMD,
angiogenesis may subside during its natural course. Interestingly,
active AMD tended to have a lower vasohibin-to-VEGF ratio, whereas
inactive AMD had a higher vasohibin-to-VEGF ratio [38]. These
observations suggest that the expression level of VASH1 may
determine certain pathological condition.
When added exogenously, VASH1 inhibits migration and
proliferation of ECs, and inhibits angiogenesis. The receptor for
vasohibin and its intracellular signaling pathways are now under
investigation. Even so, one may ask how exogenous VASH1 can
exhibit its effect on angiogenesis in the presence of endogenous
VASH1. Our recent analysis clarified that exogenous
VASH1 exhibited little effect in the termination zone where
endogenous vasohibin was present, but effectively inhibited
angiogenesis in the sprouting zone where endogenous VASH1 was
not present [35] (figure
2). Since exogenous VASH1 can efficiently inhibit
angiogenesis, one may anticipate the application of VASH1 in
antiangiogenic therapy. So far, we have been able to show the
effect of VASH1 on at least three different states of
pathological angiogenesis; tumor angiogenesis, arterial adventitial
angiogenesis related to atherosclerosis and ocular angiogenesis
[29, 36, 41, 42].
Conclusion
The present mini-present review focuses on two angiogenesis
inhibitors, Dll4 and VASH1, produced by ECs. Accumulating
evidence indicates that the spacio-temporal expression patterns and
roles of these two angiogenesis inhibitors are distinct.
Dll4 is expressed in tip and some stalk cells and determines
the number of sprouts (figure 1), whereas
VASH1 is expressed in ECs in the termination zone and
determines the termination of angiogenesis (figure 2).
Recently, several other angiogenesis inhibitors have also been
reported to be expressed in ECs. Netrin family members were
originally identified as a regulator of axon guidance. Among them,
netrin-4 was recently shown to be induced in ECs by VEGF
stimulation, and to inhibit angiogenesis via binding to neogenin
and recruitment of Unc5B [43]. However, since the spacio-temporal
expression pattern of netrin-4 is not known, the precise role
of netrin-4 in the regulation of angiogenesis remains to be
elucidated. Nevertheless, we propose that angiogenesis inhibitors
produced by ECs orchestrate and regulate angiogenesis in a
complementary manner.
Another issue is the balance of angiogenesis stimulators and
inhibitors. The original idea of this balance theory is based on
the scenario that angiogenesis is initiated when stimulators are
up-regulated and inhibitors are down-regulated [44]. This theory is
derived from the idea that angiogenesis inhibitors act as barriers
of angiogenesis. However, the scenario is not so simple, as some
angiogenesis inhibitors are up-regulated in ECs during
angiogenesis, and finely tune this process. Clearly, this theory
needs to be re-evaluated depending on the individual
inhibitors.
Acknowledgments
This author is supported by a Grant-in-Aid for Scientific Research
on Priority Areas from the Japanese Ministry of Education, Science,
Sports and Culture, and by Health and Labour Sciences research
grants, Third Term Comprehensive Control Research for Cancer, from
the Japanese Ministry of Health, Labour, and Welfare.
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