Editorial
Neointima Formation
A Local Affair
Virginia J. Hoglund, Xiu Rong Dong, Mark W. Majesky
W
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here do intimal smooth muscle cells (SMCs) come
from? For many years, the idea that intimal SMCs
originated from the underlying media went unchallenged.1
Then, reports2,3 began to appear that up to half of the SMCs
in the intima of atherosclerotic plaques and injured arteries
arose from circulating progenitor cells of bone marrow origin.
This new view of intimal SMC formation was potentially
important because it raised the possibility of a new class of
therapeutic targets for intervention in the process of restenosis based on a bone marrow derivation of intimal SMCs.
However, as other laboratories began to follow up on these
intriguing initial reports, a long-term contribution of bone
marrow– derived cells to intimal tissue became less tenable.4,5
In this issue of Arteriosclerosis, Thrombosis, and Vascular
Biology, a careful and detailed study by Daniel et al6 seems to
leave little or no room for a role of bone marrow– derived
cells as progenitors for the intimal SMCs and endothelial
cells that are stable residents of a mature neointima that forms
after acute vascular injury.
teins calponin or SM myosin heavy chain (Figure). Likewise,
few, if any, of the CD31pos endothelial cells in the neointima
could be assigned a bone marrow origin by 16 weeks after
injury. The researchers6 conclude that the contribution of
marrow-derived cells to neointimal formation is best viewed
as an early paracrine activity that diminishes with time and
that there is little, if any, long-term contribution of marrow-
See accompanying article on page 1890
Long-Term Analysis of Neointimal Formation
Daniel et al6 transplanted bone marrow from enhanced green
fluorescent protein (EGFP)–positive (EGFPpos) mice into
lethally irradiated wild-type C57Bl/6 mice, allowed 12 weeks
for stable engraftment, performed wire injury to the femoral
artery, and then recovered injured tissues from 3 days to 16
weeks after injury. The extended time course is important
because most studies of neointimal formation in this model
usually stop at 4 weeks (occasionally at 8 weeks) after
vascular injury. Daniel et al observed rapid accumulation of
EGFPpos cells in neointimal tissue that peaked in the first 2
weeks after injury and accounted for up to 68% of the total
cells in the neointima. However, as cells expressing differentiated SMC markers began to accumulate in the neointima,
the number of EGFPpos cells in the neointima became
markedly diminished. By 16 weeks after injury, only 2% of
neointimal cells were EGFPpos and few, if any, EGFPpos
neointimal cells expressed the definitive SMC marker proFrom the Seattle Children’s Research Institute (V.J.H., X.R.D., and
M.W.M.), University of Washington, Seattle; the Departments of Pediatrics and Pathology (M.W.M.), University of Washington, Seattle; and
Curriculum in Genetics and Molecular Biology, Department of Genetics
(V.J.H. and M.W.M.), University of North Carolina, Chapel Hill.
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]
(Arterioscler Thromb Vasc Biol. 2010;30:1877-1879.)
© 2010 American Heart Association, Inc.
Figure. Bone marrow– derived cells are not stable residents in
the neointima. A, Chimeric mice transplanted with EGFPexpressing bone marrow cells are subjected to femoral artery
injury. B, Two weeks after wire injury, the neointima is composed of MoMa2⫹ EGFP⫹ macrophages and Sca1⫹ CD34⫹
EGFP⫺ vascular progenitor cells. C, Sixteen weeks after injury,
there are few, if any, EGFP⫹ cells in the neointima. Many of the
neointimal SMCs in this model originate from locally derived
adventitial Sca1⫹ CD34⫹ EGFP⫺ cells (AdvSca1). EEL indicates
external elastic lamina; IEL, internal elastic lamina.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.110.211433
1877
1878
Arterioscler Thromb Vasc Biol
October 2010
derived progenitors to the endothelial or SMC components of
the long-standing neointima.
Experimental Design and
Technical Considerations
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Several technical and experimental considerations are important in evaluating why the results of Daniel et al6 reach
conclusions that significantly differ from those of earlier
reports.2,3 One is the use of high-resolution confocal microscopy and deconvolution analysis of z-axis image stacks.
These methods reduce the likelihood of false-positive assignments for EGFPpos cells that appear to express SMC marker
proteins that are actually expressed by closely apposed cells
in tissue cross sections. A second technical consideration is
that Daniel et al found that rapid fixation with 4% paraformaldehyde was necessary to prevent leakage of the EGFP
marker protein from a cell that expressed it in vivo to a
neighboring cell that did not.6 If too much time was allowed
before fixation, cells in the tissue began to release EGFP,
most likely through cell lysis, and the fluorescent marker
diffused to neighboring binding sites associated with otherwise EGFP-negative (EGFPneg) cells. In addition, an important feature of their experimental design was that the time
course was extended up to 16 weeks after arterial injury. This
extended time course allowed for a clear distinction between
an early inflammatory phase occurring in the first 2 weeks
after injury (when the neointima contained a large component
of EGFPpos/monocyte ⫹ macrophage-2 (MoMa2)pos macrophages) and a late mature phase from 4 to 16 weeks (when
EGFPpos cells were lost and EGFPneg cells expressing SM
␣-actin and the definitive SMC marker proteins [calponin and
SM myosin heavy chain] became abundant) (Figure). The
results of the study by Daniel et al strongly suggest that the
role of bone marrow– derived cells in neointimal formation is
primarily a transient paracrine one rather than as a precursor
for transdifferentiation into long-term resident endothelial
cells and SMCs in neointimal tissue.
Origins of Neointimal SMCs
What then is the origin of SMCs in the long-standing
neointima? In simpler times, the finding that bone marrow–
derived progenitors do not serve as a significant source of
neointimal SMCs would lead to the familiar conclusion that
medial SMCs are the source of intimal SMCs. Except in
extreme models of transplant graft rejection, in which essentially all of the medial cells in the graft are lost through cell
death, there is little argument that at least some neointimal
SMCs originate in the injured media. However, the media is
not the only source of cells from which the injured artery wall
can build a neointima. Over the years, there have been
occasional reports about a possible role for the adventitia in
neointimal formation. For many investigators, these reports
were seen as inconsistent with existing dogma and their
conclusions were frequently ignored. However, it is hard to
deny that the adventitia is highly responsive to most forms of
arterial injury. Indeed, there appears to be a continuous
communication between the endothelium and the adventitia
via transmural mediators whose molecular identity has not
yet been characterized. For example, Scott et al7 reported that
most proliferating cells in porcine coronary arteries subjected
to overstretch injury were found in the adventitia. Injections
of bromodeoxyuridine given between days 2 and 3 after
injury showed that proliferating adventitial cells can migrate
into the neointima, where they were found by day 14 after
injury.7 Similar results were reported by Shi et al8 using a
saphenous vein graft model. Likewise, in a rat carotid balloon
injury model in which adventitial fibroblasts are labeled in
vitro with a retrovirus expressing ␤-galactosidase and then
introduced into the carotid adventitia immediately after injury, ␤-galactosidase–positive cells were found in the injured
media at 5 days and in the neointima at 7, 10, and 14 days
after injury.9 A review of the literature in 2001 led Sartore et
al10 to hypothesize that in response to adult vascular injury,
activation of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that invests medial
SMCs around newly forming blood vessels.
Formation of Adventitia in
Vascular Development
The suggestion of a link between mechanisms used to
construct artery walls in the embryo and the formation of a
neointima in adult vessels raises a number of intriguing
questions, particularly with respect to the adventitia. It is well
documented that hypertensive remodeling of the hypoxic
pulmonary artery results in dramatic wall thickening, including the formation of additional smooth muscle layers. These
additional layers form on the adventitial side of the artery
wall and may well use SMCs that originate from local
progenitors that are natural residents in the adventitia.11
Similarly, in mice haploinsufficient for tropoelastin, additional alternating layers of SMCs and elastic fibers are
formed on the adventitial side of the artery wall during the
late stages of embryogenesis (around E15.5 to E18.5).12,13
These new SMCs are formed well after the initial cohort of
SMC progenitors from the cardiac neural crest and other early
embryonic sources have completed investment of artery walls
from E10.5 to E14.5.14 A potential source of SMCs that are
added to the outside of the artery wall was identified by Hu
et al15 and confirmed by Passman et al.16 These studies
showed that a population of stem cell antigen 1 (Sca1)–
positive cells in the adventitia (AdvSca1) could be isolated by
immunomagnetic selection or fluorescence-activated cell
sorting for Sca1 expression and found that a significant
fraction of these progenitor cells differentiated into SMCs in
vitro. These AdvSca1 progenitor cells clustered in the border
region between the media and adventitia.16 When transplanted to the outside of a vein graft, AdvSca1 cells were
found in the media at 2 weeks and in the neointima at 4 weeks
after transplantation, where they composed approximately
20% of the total neointimal cell population.15 AdvSca1 cells
normally appear at approximately E16.5 to E17.5 in the aortic
adventitia and then are found throughout the arterial system.16
They are not derived from the bone marrow.15 Their initial
appearance and/or survival in the embryonic aortic adventitia
is dependent on sonic hedgehog signaling in the adventitial
layer.16 In this same border region between the media and
adventitia of human internal thoracic artery specimens, a
population of CD34pos/platelet-endothelial cell adhesion
Hoglund et al
molecule-1 (PECAM-1)neg cells is found; this population can
form capillary-like microvessels when segments of human
internal thoracic arteries are explanted in an aortic ring
assay.17
Summary
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Taken together, these reports suggest that the arterial adventitia contains a resident population of vascular progenitor
cells with the capability to differentiate into SMCs and to
migrate from the adventitia to the developing neointima.
Indeed, Daniel et al6 identify a highly proliferative fraction of
Sca1pos/CD34pos cells in the femoral artery adventitia that
was EGFPneg and, therefore, locally derived. Moreover, at 6
weeks after wire injury, individual Sca1pos/CD34pos cells
were found within the media, apparently migrating from the
adventitia to the neointima, similar to the findings of Hu et
al.15 The study by Daniel et al6 provides further support for
the conclusions of Wagers et al18 that bone marrow– derived
hematopoietic stem cells exhibit little or no plasticity for
transdifferentiation into the principle cell types that make up
the vessel wall (endothelial cells and SMCs). The unique
properties of intimal SMCs argue for a unique origin for these
cells.19 The studies of Bentzon et al4 and Daniel et al, among
those of other researchers, suggest that an origin of intimal
SMCs from circulating progenitors of bone marrow origin is
highly unlikely. Persistent reports of adventitial cell proliferation after arterial injury,6,7 the movement of these cells into
the intima,7,9,15 and the formation of a vascular progenitor cell
niche at the border between the media and the adventitia in
embryonic development15–17 argue for a closer look at the
roles played by adventitial cells in the formation of a
neointima and in the pathogenesis of intimal disease.
Acknowledgments
We thank our colleagues Jenna Regan, PhD, and James Faber, PhD,
at the University of North Carolina; and Alexander W. Clowes, MD,
Mary Weiser-Evans, PhD, Joseph M. Miano, PhD, and Stephen M.
Schwartz, MD, PhD, for their helpful discussions.
Sources of Funding
This study was supported by American Heart Association Fellowship
09PRE2060165 (Hoglund); grants HL-93594 and HL-19242 from
the National Institutes of Health (Dr Majesky); and the Curriculum
in Genetics and Molecular Biology Training Program, University of
North Carolina, Chapel Hill.
Disclosures
None.
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KEY WORDS: restenosis
䡲 smooth muscle cell 䡲 adventitia 䡲 bone marrow
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Neointima Formation: A Local Affair
Virginia J. Hoglund, Xiu Rong Dong and Mark W. Majesky
Arterioscler Thromb Vasc Biol. 2010;30:1877-1879
doi: 10.1161/ATVBAHA.110.211433
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