Editorial
Patterning the Artery Wall by Lateral Induction of
Notch Signaling
Virginia J. Hoglund, BS; Mark W. Majesky, PhD
B
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creased (1.22-fold), the wall was thicker (1.24-fold), and
the number of smooth muscle cells (SMCs) in the tunica
media was increased by 30%.4 Despite these increases in
overall wall size, there were no changes in the number of
layers in any of the arteries examined in MtGH transgenic
mice. Thus, the aortas of MtGH transgenic mice were able
to compensate for additional demands for cardiac output
and the consequent increases in wall tension necessary to
support a larger animal by increasing the number and size
of SMCs found within thicker individual layers, but the
number of SMC-containing layers seemed to be fixed.
Because the increases in circulating growth hormone in
MtGH transgenic mice become apparent after birth, these
findings pointed to mechanisms that operate in utero to
determine the characteristic number of layers in the tunica
media that are found in different arteries in adult animals.
More recently, a second transgenic model was described
that confirmed and extended this conclusion. Mice that are
completely deficient in elastin (Eln⫺/⫺) die of obstructive
arterial disease in the neonatal period.5 However, Eln⫹/⫺
mice survive and exhibit various structural adaptations in
their elastic arteries.6 The walls of the ascending aorta,
common carotid artery, and abdominal aorta are thinner,
and these arteries are smaller in overall diameter than in
the wild-type littermates. Yet, Eln⫹/⫺ mice exhibit an
increase of ⬇35% in the number of layers of elastic
lamellae in thoracic and abdominal aorta.6,7 The extra
layers begin to appear at approximately embryonic day
18.5 (E18.5) and are completed by birth.6,7 Because the
normal number of smooth muscle-containing layers in the
aorta is laid down by E14.5 to E15.5, the Eln⫹/⫺ mice
would appear to define a second window of time from
E18.5 to birth in which the artery wall can adjust the
number of smooth muscle layers to normalize wall strain,
but after birth this adaptive mechanism appears to no
longer operate.
lood vessels exhibit a common structure composed of
layers of cells encircling a central lumen. Arteries
generally have more layers than veins have, and different
arteries in the same individual can have different numbers
of layers. Because all blood vessels are built around a
monolayer of endothelial cells, the variation in wall
structure is due to variable numbers of smooth musclecontaining layers in the tunica media. Indeed, Wolinsky
and Glagov’s classic article showed that the relation
between lumen diameter and wall thickness across a wide
range of mammalian species is a function of the number of
layers of smooth muscle and elastic fibers that are present
in the arterial media.1 Yet precisely how layers of smooth
muscle are formed during vascular development and what
molecular mechanisms operate to produce different numbers of layers in different blood vessels is still poorly
understood.
Article see p 314
Lessons from Transgenic Mice
For many years the prevailing hypothesis has been that
wall thickness is determined by wall tension, and the
number of layers found in a given blood vessel is that
number sufficient to normalize wall tension to values
(dynes/cm2) within a narrow physiological range per
layer.2 The advent of transgenic mouse technology generated new animal models that provided for interesting tests
of the accepted paradigms for the development of vessel
wall structure. For example, mice expressing a growth
hormone transgene driven by a metallothionein promoter
(MtGH) reached a body mass 1.8-fold that of wild-type
littermate mice.3 Most of the internal organs in these mice
exhibited proportional increases in mass in comparison
with wild-type littermates (eg, kidney⫽1.9-fold; heart⫽
1.8-fold).4 To accommodate the increases in blood flow
required to support these larger bodies, the aortas of
transgenic MtGH mice exhibited a number of structural
changes. For example, aortic lumen diameter was in-
The Role of Notch Signaling in Layer
Formation in Developing Arteries
Recently, evidence has been accumulating to suggest that
at least one important mechanism that functions to organize and pattern SMCs in artery walls is Notch signaling.
The earliest steps in formation of the tunica media involve
establishment of adhesive contacts between endothelial
cells and smooth muscle progenitors via heterotypic cellcell adhesion mediated by N-cadherin.8 This brings endothelial cell surface molecules into contact with partners on
adjacent SMC progenitors, triggering SMC differentiation
in the progenitors and the initiation of tunica media
formation. With continued development, an iterative se-
The opinions expressed in this article are not necessarily those of the
editors or of the American Heart Association.
From the Seattle Children’s Research Institute (V.J.H.), Departments of Pediatrics and Pathology (M.W.M.), and The Institute of
Stem Cell & Regenerative Medicine (M.W.M.), University of Washington, Seattle, WA.
Correspondence to Mark W. Majesky, PhD, Seattle Children’s Research Institute, University of Washington, 1900 Ninth Ave, M/S C9S-5,
Seattle, WA 98101. E-mail [email protected]
(Circulation. 2012;125:212-215.)
© 2011 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.111.075937
212
Hoglund and Majesky
Lateral Induction of Notch Signaling
213
quence is set into motion that assembles SMC progenitors
into the number of layers characteristic for a given artery.1
This process appears to involve physical contact between
incoming cells and cells already present in the developing
vessel wall.8,9 Notch signaling is initiated by 4 Notch
receptors (Notch 1– 4) interacting with 5 Notch ligands
(Jagged 1, 2 and Delta-like 1, 3, and 4).10 –12 Both the
ligands and receptors are transmembrane proteins whose
activation is promoted by cell-cell contact. Evidence that
Notch signaling plays an important role in endothelial
contact-dependent differentiation of smooth muscle progenitor cells in vivo was provided by the results of
cell-specific deletion of Notch signaling activity in neural
crest-derived progenitors13 and in experiments where the
Notch ligand Jagged-1 (Jag1) was deleted specifically in
endothelial cells.14
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Positive Feedback Regulation of Jag1
Expression in Smooth Muscle Cells
As a further test of the hypothesis that Notch mediates
patterning of the artery wall, Manderfield et al report in
this issue of Circulation that expression of dominantnegative mastermind-like protein (DN-MAML), an inhibitor of target gene activation by all forms of Notch
receptor,15 produced significant reductions in Jag1 expression in neural crest-derived SMCs surrounding paired
aortic arch arteries.16 Moreover, when E17.5 aortic SMCs
were plated on a substrate that contained immobilized
Jag1-Fc fragments that are known to activate Notch
receptors, an increase in expression of Jag1 was observed,
suggesting a positive feedback regulation of Jag1 expression by these cells. Evidence supporting the idea that
Notch signaling directly regulates Jag1 expression in aortic
SMCs was obtained by identification of an evolutionarily
conserved region (ECR) in intron 2 of the Jag1 locus that
bound both Rbp-j/CSL and Notch ICD and drove expression of a reporter gene in a Notch-responsive fashion in
cultured SMCs. This genomic element, termed ECR6, was
then examined in transient transgenic mice and found to be
sufficient to direct LacZ reporter gene expression in a
temporal and spatial pattern that closely matched that of
the endogenous Jag1 gene.16 Interestingly, the ECR6
reporter transgene in stable transgenics exhibited robust
activity in SMCs derived from the cardiac neural crest, but
was not active in descending aortic SMCs that originate
from paraxial mesoderm.17–19 Finally, loss of Jag1 expression in developing neural crest-derived aortic SMCs produced congenital heart defects consisting of abnormal
aortic arch artery patterning, reduced expression of the
canonical Notch target gene Hrt1, and reduced expression
of SMC differentiation markers.16
Role of Lateral Induction in Assembly of the
Artery Wall
Therefore, the current evidence strongly suggests that
endothelial cells of developing aortic arch arteries express
Jag1 on their cell surface that then engages a Notch
receptor on adjacent neural crest-derived SMC progenitor
cells to promote differentiation and assembly of the first
Figure. Artery wall formation begins with the interaction of jagged1 (Jag1)-expressing endothelial cells with mural cell progenitors expressing Notch receptors (Notch). This interaction both
promotes smooth muscle differentiation and upregulates expression of Jag1 in the newly formed smooth muscle cells, thus initiating a feed-forward pathway in the nascent medial layer (Layer
1). Jag1 expressed by cells in Layer 1 engages Notch receptors
in surrounding mural cell progenitors leading to smooth muscle
differentiation and upregulation of Jag1 expression in mural cells
that will go on to form Layer 2. This positive feedback response
of Jag1 induction by Notch signaling in the developing artery
wall is called lateral induction. By this sequential lateral induction process, a multilayered artery wall is formed.
layer of the tunica media. Results presented by Manderfield et al,16 and by Feng et al20 and Liu et al,21 as well,
argue that the initial signal is then propagated to the next
layer of artery wall by lateral induction of Jag1 expression
in newly differentiated SMCs (Figure). This simple feedforward pathway is an elegant switch acting sequentially to
turn on SMC differentiation and assemble the next layer of
the media in a repeating pattern. Yet, for a more complete
understanding of this important process of artery wall
assembly and patterning, a number of remaining questions
will need to be addressed in future studies.
Remaining Questions
1. We know that individual layers in the arterial media
are composed of more than SMCs alone. Indeed the
term elastic lamellae is often used to describe these
layers, a reference to the abundance of cross-linked
elastin, fibrillin, lysyl oxidase, and elastic fiber
proteins that also alternate in layers across the artery
wall.7 Does Notch signaling by lateral induction also
drive expression of genes encoding tropoelastin and
the multiple elastic fiber proteins that are required to
214
2.
3.
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4.
5.
6.
7.
Circulation
January 17, 2012
make a mature layer of artery wall? If so, is this a
direct effect of Notch signaling on individual matrix
protein gene regulatory elements, or is it an indirect
effect of triggering SMC differentiation in appropriate progenitor cells?
How is lateral induction of Notch signaling coordinated with other signals that are important for artery
wall formation, such as transforming growth
factor- ␤ , bone morphogenetic protein, and
myocardin-related transcription factor B?22
The genomic regulatory element identified here,
ECR6, mediates lateral induction of Jag1 expression
in neural crest-derived progenitors during SMC differentiation. Yet ECR6 does not seem to have this
activity for SMCs that differentiate from non–neural
crest progenitors.16 Because the structure of the
artery wall with respect to layers of SMCs and elastic
fibers is not obviously different in arterial segments
of non–neural crest origin, does lateral induction of
Notch signaling also explain layer formation in these
non–neural crest arterial segments? If so, what
genomic elements are responsive to lateral induction
in the Jag1 locus in non–neural crest-derived SMC
progenitor cells? If not, then what signaling pathway(s) mediate smooth muscle layer formation in
non–neural crest-derived arteries?
What molecular mechanisms become active to produce more elastic layers in aortas of Eln⫹/⫺mice?5,6,23
Is Notch signaling by lateral induction reactivated
late in artery wall development (⬇E18.5) to mediate
formation of an ectopic series of elastic lamellae in
Eln⫹/⫺ aorta?5–7
Given the consistently observed correlation between
the physical forces producing wall tension and the
structural component of wall thickness in many
different arteries, is lateral induction of Notch signaling responsive to changes in wall tension during
vascular development? If so, what is the biochemical
nature of the coupling mechanism?
If all arteries begin wall formation in the same
way— endothelial expression of Jag1 activates Notch
signaling in SMC progenitors leading to Jag1 expression, etc—what mechanisms terminate the iterative
layer sequence to produce arteries with different
numbers of SMC layers in their tunica media? Do
morphogenic pathways associated with development
of the tunica adventitia, such as sonic hedgehog
signaling,24 act to terminate smooth muscle layer
formation, or does the lateral induction signal decay
at some point, thus ending the repeating sequence?
Although mammals produce artery walls with SMCs
as essentially the only cell type within elastic lamellae, other species including birds and reptiles produce
alternating layers of SMCs and non-SMCs (called
interlaminar cells) within the media of large elastic
arteries.25,26 Does lateral induction of Notch signaling
also operate to build a multilayered artery wall in
these phylogenetically distinct cases as well?
Summary
Lateral induction of Notch signaling is an attractive mechanism to play an important role in building a multilayered
artery wall. Such a role is consistent with Jag1 loss-offunction phenotypes in mice14,20 and in patients with Alagille
syndrome where haploinsufficiency for Jag1 leads to congenital heart disease and mispatterning of the great vessels.27,28
Although many questions remain to be answered about
morphogenesis of the tunica media, pursuit of Notch signaling through Jag1 in SMC progenitors promises to be a very
fruitful way forward.
Sources of Funding
This work was supported by National Institutes of Health grants
HL93594 and HL19242 (to M.W.M.), American Heart Association
grant 09PRE2060165 (to V.J.H.), the Curriculum in Genetics and
Molecular Biology at the University of North Carolina at Chapel
Hill, and the Seattle Children’s Research Institute.
Disclosures
None.
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KEY WORDS: Editorials 䡲 vascular development
䡲 Notch signaling 䡲 neural crest
䡲 smooth muscle progenitor cell
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Patterning the Artery Wall by Lateral Induction of Notch Signaling
Virginia J. Hoglund and Mark W. Majesky
Circulation. 2012;125:212-215; originally published online December 6, 2011;
doi: 10.1161/CIRCULATIONAHA.111.075937
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