ATVB In Focus
Smooth Muscle Cells
Series Editor:
Giulio Gabbiani
Origin of Vascular Smooth Muscle Cells and the Role of
Circulating Stem Cells in Transplant Arteriosclerosis
Jan-Luuk Hillebrands, Flip A. Klatter, Jan Rozing
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Abstract—To date, clinical solid-organ transplantation has not achieved its goals as a long-term treatment for patients with
end-stage organ failure. Development of so-called chronic transplant dysfunction (CTD) is now recognized as the
predominant cause of allograft loss long term (after the first postoperative year) after transplantation. CTD has the
remarkable histological feature that the luminal areas of intragraft arteries become obliterated, predominantly with
vascular smooth muscle cells (VSMCs) intermingled with some inflammatory cells (transplant arteriosclerosis, or TA).
The development of TA is a multifactorial process, and many risk factors have been identified. However, the precise
pathogenetic mechanisms leading to TA are largely unknown and, as a result, adequate prevention and treatment
protocols are still lacking. This review discusses the origin (donor versus recipient, bone marrow versus nonbone
marrow) of the VSMCs in TA lesions. Poorly controlled influx and subsequent proliferative behavior of these VSMCs
are considered to be critical elements in the development of TA. Available data show heterogeneity when analyzing the
origin of neointimal VSMCs in various transplant models and species, indicating the existence of multiple sites of origin.
Based on these findings, a model considering plasticity of VSMC origin in TA in relation to severity and extent of graft
damage is proposed. (Arterioscler Thromb Vasc Biol. 2003;23:380-387.)
Key Words: chronic transplant dysfunction 䡲 transplantation 䡲 transplant arteriosclerosis
䡲 origin 䡲 vascular smooth muscle cells
success has steadily improved or remained at the same level
as in the precyclosporin A era and so far, no new drugs are
available that can further extend graft survival time. CTD is
now recognized as the primary cause of allograft loss after the
first year after transplantation.2
Solid-Organ Transplantation and Chronic
Transplant Dysfunction (CTD)
Since the late 1950s, transplantation of solid organs has
become an increasingly successful therapy for patients suffering from end-stage organ failure. The short-term results of
organ transplantation have significantly improved over recent
decades. This improvement is primarily because of the
introduction of new, more effective immunosuppressive
agents. Moreover, improved histocompatibility leukocyte antigen tissue-typing assays and surgical techniques as well as
advances made in donor organ preservation contributed to the
decreased morbidity and mortality after solid-organ transplantation. Despite the use of these new drugs, however, it
has become clear that clinical transplantation has not
achieved its goals as a long-term treatment. Although a steady
improvement in the long-term survival of renal grafts since
the late 1980s has been described,1 this effect is less clear in
other organs. Depending on the organ grafted, long-term
See page 379
CTD can be defined clinically as the progressive irreversible loss of graft function that occurs late in the posttransplant period (months to years after transplantation).3 The
incidence of CTD after transplantation depends on the type of
organ grafted and varies from 3% in liver allografts to ⬎70%
in lung allografts 5 years after transplantation.4 – 6 The clinical
presentation of CTD generally implies deterioration of graft
function and is associated with a variety of organ-specific
clinical parameters.7 In kidney transplants, CTD is characterized among others by decreased glomerular filtration rate,
increased levels of plasma creatinin, and proteinuria. CTD in
Received October 1, 2002; revision accepted December 18, 2002.
From the Department of Cell Biology, Section Immunology, Faculty of Medical Sciences, University of Groningen, The Netherlands.
Correspondence to J.L. Hillebrands, PhD, Department Cell Biology/Section Immunology, Faculty of Medical Sciences, University of Groningen, A.
Deusinglaan 1, NL-9713 AV Groningen, The Netherlands. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000059337.60393.64
380
Hillebrands et al
VSMCs and Stem Cells in Transplant Arteriosclerosis
381
Figure 1. TA in a rat aortic allograft model. Double staining for
vascular SMA and elastin. A, Healthy normal aorta. B, Aorta
showing severe TA with extensive neointimal hyperplasia. A
indicates tunica adventitia; IEL, internal elastic lamina; M, tunica
media; NI, neo-intima.
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cardiac allografts is characterized by increased frequency of
myocardial infarction, arrhythmia, and sudden death. Finally,
lung and liver transplant recipients suffering from CTD
present with decreased pulmonary function and increased
levels of bilirubin and liver enzymes in blood.4,7,8 The
histopathological characteristics associated with CTD also
vary between the different organs.7,9 Kidney allografts are
characterized by glomerular sclerosis and tubular atrophy,
whereas cardiac transplants with CTD presents with parenchymal fibrosis. However, lung and liver transplants with
CTD are characterized by the presence of obliterated bronchioli and degeneration of the bile ducts, respectively.4,7–10
However, besides graft-specific histopathology, there is also
a common histomorphological feature of CTD, particularly in
kidney and heart transplants, that also can be observed in liver
and lung allografts: transplant arteriosclerosis (TA).9 –13
Transplant Arteriosclerosis (TA)
TA is characterized by vascular lesions in the graft that
consist of concentric myointimal proliferation, resulting in
the development of an occlusive neointima (NI) in the arterial
structures of the graft.3 The NI primarily consists of smooth
muscle ␣-actin (SMA)-positive vascular smooth muscle cells
(VSMCs; Figure 1), which are believed to be derived from
the vessel media.4,10 This progressive blood vessel occlusion
could lead to downstream ischemic tissue damage and disruptive fibrosis and has therefore generally been accepted as
the main cause of progressive deterioration in graft function
(CTD). Other findings coinciding with TA include a persistent focal perivascular inflammation (perivasculitis), endothelial swelling and inflammation (endothelialitis), disruption of
the internal elastic lamina, thinning of the vascular media
(loss of medial SMA-positive VSMCs), foam cell accumulation in the NI, and presence of macrophages and T cells in the
neointimal lesion.14 In contrast with ordinary atherosclerosis,
which is usually focal and eccentric, the common form of TA
is concentric and generalized.
The presence of persistent perivascular inflammation in TA
suggests that alloresponse-mediated injury of the graft vessels is
the prime cause of TA development. However, the etiology of
TA remains poorly defined. The pathogenesis of TA seems to be
multifactorial, and although alloreactivity of the host against the
Figure 2. Current paradigm on the development of intimal
hyperplasia in transplant arteriosclerosis: neointimal VSMCs
originate from the graft: donor-derived.
graft appears to be the most important factor contributing to the
development of TA, alloantigen-independent factors also seem
to be associated with the pathogenesis of TA.15
Despite discrepancies in histopathology between ordinary
atherosclerosis and TA, the “response-to-injury” paradigm
applicable to atherosclerosis and originally proposed by
Ross16 has been accepted widely for the development of TA.
This paradigm proposes that transplant-related trauma (alloantigen dependent and independent) causes activation and
damage of endothelial cells (ECs) along the graft arterial
system. The damaged and activated endothelium subsequently initiates a generalized remodeling process that is
coordinated by proinflammatory cytokines and growth factors produced by the activated ECs themselves as well as
vessel wall parenchymal cells and inflammatory cells. Moreover, the immune response characterized by perivascular
inflammation induces further low-grade damage to the vascular endothelium. This cascade of events eventually results
in replication of VSMCs in the vascular wall, influx of
VSMCs into the subendothelial space (intima), and generation of the neointimal lesion.4
Origin of VSMCs in TA
Current thinking on the process of TA, as described above,
holds that in response to cytokines, growth factors, and other
inflammatory mediators produced by inflammatory cells and
damaged/activated graft endothelium, donor-derived medial
VSMCs of affected arteries start to proliferate and migrate
from the media into the subendothelial space just beneath the
EC layer.9 During this process, they transform their phenotype from “contractile” to “synthetic” and become capable of
replication.16 According to this concept, neointimal VSMCs
in TA originate from graft tissue and therefore should be
donor derived (Figure 2).
If neointimal VSMCs do indeed originate from the donor,
they should be demonstrably graft derived. As long ago as the
early 1960s, Woodruff17 and Medawar18 proposed that replace-
382
TABLE 1.
Arterioscler Thromb Vasc Biol.
March 2003
Origin of Intimal Cells in Various Models
Species
Model
Organ
Cell Type
Cell Source
Prevalence, %
Identification
Reference
Human
Allo Tx in sex-mismatched
combinations
Heart
VSMC, EC
Recipient-type
⬍5
Y-probe
20
Human
Allo Tx in sex-mismatched
combinations
Heart
VSMC
Recipient-type
5 to 10
Y-probe/SMA
27
Human
Allo Tx in sex-mismatched
combinations
Heart
VSMC
EC
Recipient-derived
Recipient-derived
60
42
Y-probe/SMA
Y-probe/factor VIII
28
Human
Allo Tx in sex-mismatched
combinations
Kidney
VSMC
Recipient-type
80 to 90
Y-probe/SMA
29
Rat
Allo Tx
Femoral artery
VSMC
Recipient-derived
100
MHC I
32
Rat
Allo Tx in sex-mismatched
rats
Heart
VSMC
EC
Recipient-derived
Donor-derived
⬎95
⬎95
Y-PCR/SMA
MHC I/HIS52
21,22
Rat
Allo Tx in sex-mismatched
rats
Aorta
VSMC
EC
Recipient-derived
Recipient-derived
⬎95
⬎95
Y-PCR/SMA
MHC I/HIS52
22
Rat
Allo Tx in BM chimeric rats
Aorta
EC
Recipient non-BM–derived
⬎95
MHC I/HIS52
26
Recipient BM-derived
⬍5
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Rat
Allo Tx in BM chimeric rats
Aorta
VSMC
Recipient non-BM–derived
⬎95
MHC I/SMA
Present study
Mouse
Allo Tx after anti-CD4 and
anti-CD8 treatment
Heart
mVSMC, EC
Donor-derived
ND
MHC II
33
Mouse
Allo Tx in sex-mismatched/
BM chimeric mice
Aorta
VSMC
Recipient non-BM–derived
⬎95
Y-probe/SMA
34
Mouse
Vein Tx in
sex-mismatched/BM
chimeric/Tg mice
Vein
VSMC
Donor-derived
Recipient non-BM–derived
60
40
LacZ/Y-probe/SMA
67
Mouse
Allo Tx in BM chimeric/Tg
mice
Heart
VSMC
Recipient-derived
BM-derived
⬇88
⬇82*
LacZ/GFP/SMA
36,38
Mouse
Allo Tx in sex
mismatched/Tg mice
Heart
VSMC
Recipient-derived
Majority
LacZ/Y-probe/SMA
35
Mouse
Allo Tx in BM chimeric/Tg
mice
Aorta
VSMC
Recipient-derived
BM-derived
⬎95
11
LacZ/SMA
37
Mouse
Induced heart ischemia
Heart
VSMC
lin⫺ c-kit⫹ BM cells
ND
GFP/SMA
62
Mouse
Induced heart ischemia
Heart
EC
BM-derived SP cells
3.3
LacZ/Flt-1
64
Mouse
Injury induced NI formation
in sex-mismatched BM
chimeric mice
Iliac artery
VSMC
BM-derived
⬇50
Y-probe/SMA
65
*Percent of neointimal cells.
Flt-1 indicates ␣-mouse-EC Mab; GFP, Green Fluorescent Protein reporter gene construct; HIS52, ␣-rat-EC Mab; mVSMC, medial-VSMC; Tg mice, transgenic mice;
Tx, transplantation; Y-PCR, male Y-chromosome specific polymerase chain reaction; Y-probe, male Y-chromosome specific probe; ND, not determined.
ment of graft endothelium with host-derived ECs might be the
reason why long-term allograft survivors experience relatively
few rejection episodes (ie, graft adaptation). Since then, several
groups have studied whether ECs covering the luminal surface
of the graft vessels after transplantation are indeed of donor
origin or had been replaced by host-derived ECs (summarized in
Table 1). Both data supporting donor19 –21 as well as recipient22–26 origin of the graft vessel endothelium have been reported, indicating heterogeneity of the underlying process.
Only recently the question has been addressed whether the
neointimal VSMCs in TA might be graft-derived (summarized in Table 1). Data indicating a donor origin of neointimal
VSMCs have been reported by Hruban et al20 Using Y
chromosome–specific fluorescence in situ hybridization on
cardiac tissue of two sex-mismatched human cardiac transplant patients, no sign of hybridization was observed in
cardiac myocytes, VSMCs, and ⬎95% of ECs, thus excluding significant replacement of transplanted cardiac tissues
with host derived cells. Glaser et al27 recently extended these
studies and found in orthotopic human cardiac transplantation
in sex-mismatched combinations approximately 5 to 10% of
VSMCs to be of recipient origin. All cardiac allografts
analyzed showed histopathological signs indicative of TA,
and the majority of host-derived VSMCs were identified in
medium and small coronary arteries. Using specific antibodies against SMA and factor VIII (to discriminate between
VSMCs and ECs, respectively), Quaini et al28 recently
reported about the presence of high levels of chimerism of
VSMCs (60%) and ECs (42%) in human cardiac allografts
transplanted in sex-mismatched combinations. Some major
differences between both studies might explain the different
outcome described in both articles. Quaini et al28 restricted
their analysis to coronary vessels with normal structure, and
areas with neointimal thickening were excluded because the
only objective was to elucidate the role of chimerism in the
undamaged myocardium. Moreover, the median graft survival
Hillebrands et al
VSMCs and Stem Cells in Transplant Arteriosclerosis
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time in their study subjects was relatively short (53 days).
However, Glaser et al27 in their study focused specifically on the
origin of VSMCs in neointimal lesions in human cardiac
allografts in which the median survival time was 5.1 years.
It seems thus from these studies in human sex-mismatched
transplants that recipient cells contribute only marginally if at
all to the expansion of neointimal VSMCs after transplantation. A role of host chimerism of VSMCs in the microvasculature (without TA) in determining graft function/survival
cannot be excluded, but this lies beyond the scope of this
review and will not be further discussed.
In contrast with human cardiac allografting, however, Grimm
and colleagues29 showed that approximately 35% of SMApositive neointimal cells in sex-mismatched renal grafts undergoing chronic rejection displayed recipient-type characteristics.
With a hybridization efficiency of 40%, this suggests that
actually 80% to 90% of neointimal SMA-positive cells were of
recipient origin. However, analysis of reciprocal combinations
clearly demonstrated also the existence of a persistent population
of donor type cells. Obviously both indications for a minimal
and for a major contribution of recipient cells in neointimal
VSMCs hyperplasia can be derived from these human studies.
Whether organ-specific differences in this process exist, which
might explain the observed differences between the cardiac and
renal transplants, remains to be elucidated and will be further
discussed below.
The question on the origin of neointimal VSMCs in TA has
recently also raised considerable interest in experimental
transplantation. Stump et al30 already in 1963 demonstrated
the existence of blood-borne VSMC precursors in experiments in which they implanted Dacron hubs in the aorta of
young pigs. These Dacron hubs became covered with ECs
and VSMC-like cells originating from cells in the blood.
Moreover, implantation of biodegradable synthetic vascular
grafts in rats showed development of new vascular wall
structures, including a neomedia (with SMA-positive
VSMCs).31 VSMCs on the Dacron hubs as well as in the
synthetic vascular grafts must by definition have been host
derived. Indeed, when Brazelton et al32 analyzed the origin of
neointimal SMCs in allografted rat femoral artery segments
using monoclonal antibodies specifically directed against
donor or recipient major histocompatibility complex (MHC)
class I antigens, all mesenchymal cells in the NI, of which
approximately 50% expressed SMA, were found to be recipient derived. Using a Y chromosome–specific single-cell
polymerase chain reaction on microdissected nuclei of SMApositive neointimal VSMCs, thereby excluding the risk of
sample contamination with infiltrating recipient-derived inflammatory cells, we showed that virtually all of the neointimal VSMCs in rat aortic and cardiac allografts are of
recipient and no longer of donor origin.21,22 These findings
have independently been confirmed in a variety of mouse
studies using MHC class II antigens,33 Y chromosome–
specific probes,34,35 and LacZ35–38 and/or green fluorescent
protein36,38 as reporter genes to define the origin of the
neointimal cells (summarized in Table 1).
There is thus now compelling evidence from experimental
studies that VSMCs in neointimal lesions after transplantation are frequently and in majority derived from recipient
383
cells and not from donor cells, which is in sharp contrast with
the current paradigm that predicts a major role for medial
VSMCs in the process of TA development.4,9,10 This finding
is actually not surprising in view of the fact that existing
medial VSMCs and associated contractility of the graft
completely disappear before (day 28 after transplantation)
vascular remodeling starts in rat aorta allografts.39 At that
time NI formation was restricted to the edges of the graft,
suggesting ingrowth of VSMC precursors from the anastomosis rather than migration of VSMCs from the graft media.
Also, Rossmann et al40 studied vascular changes in rat aortic
allografts at various times after transplantation. They observed severe destruction of the allograft media, as indicated
by karyolysis of the cells, depletion of SMA, and focal
calcification. Subsequently, intimal thickening occurred as a
result of proliferation of longitudinally oriented SMApositive myointimal cells. No signs of migration or proliferation of medial VSMCs were observed. Similar results were
reported by others.41– 43
Although recipient cells thus appear to contribute significantly to neointimal VSMCs hyperplasia in TA, it should,
however, be noted that especially rodent studies have provided high percentages of donor VSMC replacement after
transplantation, whereas in most human studies no or only
low percentages of recipient-type VSMCs could be identified
(summarized in Table 1). One of the most striking differences
between experimental murine grafts and human organ transplants displaying TA is that medial cell destruction in rodent
allografts leads to complete medial necrosis,22,39 – 43 whereas
in human allografts medial VSMC cellularity frequently
remains unchanged.42 This is probably because of the fact that
immunosuppressive treatment is not or only minimally used
in most experimental animal models that have been used to
study the origin of VSMCs in TA and this contrasts the
situation in clinical human transplantation.44 One can imagine
that when viable medial VSMCs remain available (eg, the
human situation) such medial cells can provide a source of
neointimal VSMCs, whereas in the case of complete destruction of medial VSMCs (eg, the murine situation) by definition
other resources are required. Also the positioning of the
transplanted tissue may influence the outcome of the process.
It is probably easier for local cells to colonize an interposition
graft, because they can invade transmurally or via pannus-like
growth from the sides. In this respect, it would be interesting
to study the origin of the neointimal VSMCs in a model
described by Mennander et al42, in which the adventitia of rat
aorta grafts is exposed to starch before syngeneic transplantation. In contrast with regular syngeneic grafts, these starchpretreated syngeneic transplants develop an inflammatory
reaction and display extensive intimal thickening at the site of
inflammation whereas medial VSMCs remain preserved.
Under these circumstances also a contribution of medial
VSMCs to intimal hyperplasia could well be envisaged in this
rodent model. However, our model of vascular transplant
arteriosclerosis after cardiac allotransplantation in immunomodulated rats also showed a small rim of presumably
donor-derived medial VSMCs remained even in almost completely occluded coronary arteries.45 Nevertheless, virtually
all neointimal VSMCs were found to be of recipient origin in
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Arterioscler Thromb Vasc Biol.
March 2003
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Figure 3. Origin of neointimal VSMCs after aortic allografting in
BM-chimeric rats. Confocal laser scanning microscopy of VSMC
origin after Dark-Agouty (DA) aortic allografting in LEWBM3BN
chimeric rats using the following monoclonal antibodies: A,
MN4-91-6 (DA MHC class I, red), ASM-1 (vascular SMA, green),
and DAPI (nuclei, blue). Arrow indicates remaining donor (DA)
class I MHC-positive VSMCs in the media. B, OX27 (BN MHC
class I, green), RECA-1 (ECs, red), and DAPI (nuclei, blue).
Arrow indicates recipient (BN) non-BM– derived class I MHCpositive VSMCs in the neointima. Insert represents higher magnification of these cells. Abbreviations: lu, lumen; m, tunica
media; ni, neointima.
such vessels.21,22 Similar findings were obtained using aortic
allografting in bone marrow (BM)-chimeric rats (this report).26
As shown in Figure 3A all of the neointimal VSMCs are not of
donor-type, even in the presence of remaining donor-type medial
VSMCs (red). The absence of SMA staining (green) in these
medial VSMCs, however, may indicate severe damage and loss
of viability of those cells. Another difference between rodent
models and human transplantation is the presence of existing
vascular lesions in the majority of human donor material, which
may provide the basis of further outgrowth of VSMCs during the
subsequent development of a post-transplant NI lesion.46 Such
lesions are rarely found in animal tissues used for transplantation. Taken together, although strong experimental evidence
exists favoring recipient involvement, the possibility of medial
or intimal VSMC contribution to NI development in TA cannot
be omitted.
Anatomical Origin of Recipient Derived VSMCs in
TA: Circulating Stem Cells?
The use of experimental animal models obviously also allows
to address questions on the anatomical origin of the recipient
cells involved in neointimal VSMC expansion.
For many years it was believed that vasculogenesis, the
process in which new blood vessels are formed through the
local differentiation of primitive endothelial precursors (eg,
angioblasts) only occurred during embryogenesis.47 In postnatal life the formation of new blood vessels was considered
to occur only through angiogenesis, eg, sprouting of new
vascular structures from existing vessels.48 However, recent
studies have shown that endothelial stem cells also exist in
adult life.49 –51 EC precursors have been isolated from the
bone marrow and from peripheral blood47,48 and can contribute to the formation of blood vessels.52,53 Circulating numbers
of EC precursors increase following ischemia or after GMCSF treatment54 and are CD34⫹ Flk-1⫹ AC133⫹.47,50 Reyes et
al55 recently identified a CD34⫺ vascular endothelial cadherin⫺ AC133⫹ Flk-1⫹ multipotent adult progenitor cell in
postnatal human bone marrow. When cultured in vitro with
the appropriate growth factors these cells differentiated into
CD34⫹ angioblasts. In vivo multipotent adult progenitor cell
can differentiate into ECs. Thus, BM-derived primitive precursor cells can contribute to the formation of blood vessels.
However, they contribute not only to blood vessel formation.
Although previously BM stem cells were considered to be
predominantly hematopoietic precursor cells, a variety of
reports (reviewed in the references 56,57) have now shown that
adult BM cells still hold the potential to differentiate into
many different tissues, including liver, neurons, muscle cells,
cardiomyocytes, and so on. Gradually, the concept of adult
stem cell plasticity has developed, for example, adult stem
cells, which were until now assumed to be committed to a
fixed range of progeny, can switch, once relocated and
appropriately stimulated, to a whole series of other progeny.58,59 Based on these studies transplantation of BM cells has
been used as a therapeutic procedure both in clinical and
experimental models of infarction and heart failure to see
whether this would lead to improvement of myocardial
performance.56 Direct injection of infarcted myocardium with
BM cells promoted angiogenesis, with some of the new
capillaries derived from BM.60 Injection of purified Lin⫺
c-kit⫹ cells resulted in the generation of new BM-derived ECs
and SMA-positive VSMCs.61,62 Similar results were obtained
after injection of G-CSF mobilized circulating human angioblast precursors in rat hearts after infarction.63 Strikingly,
functional improvement in these studies resulted from the
generation of new human capillaries, not through the generation of new myocytes. Infusion of LacZ-marked BM-derived
stem cells, for example, side-population (CD34⫺/low c-kit⫹
Sca-1⫹) cells, into lethally irradiated mice followed by
ischemia induction resulted in 3.3% endothelial engraftment,
indicating circulatory capacities of such cells.64 Similarly,
analysis of VSMC-like cells in irradiated sex-mismatched
BM-reconstituted Ly5-congenic mice in damage-induced
neointimal lesions showed that approximately 50% of the
neointimal SMA-positive VSMCs are of male, and thus of
BM origin.65,66 These studies convincingly show that primitive BM cells can circulate through the body and contribute to
vascular remodeling on tissue damage, giving rise to both
ECs and VSMCs. It is therefore not surprising that several
groups have addressed the issue of BM involvement in the
process of NI development and VSMC hyperplasia.
Shimizu et al37 found in a mouse aortic allotransplant
model using bone marrow chimeric LacZ-transgenic mice
that, although all neointimal VSMCs were exclusively host
derived, approximately 11% of these cells were of BM origin,
the remainder being derived from radio-resistant hostnon-BM cells that can seed the graft. Similar findings were
reported by Li et al34 Using BM-chimeric rats, allowing
discrimination between BM and non-BM– derived cells, we
also recently showed that the host-derived ECs in advanced
neointimal lesions in aortic allografts are primarily non-BM
derived.26 As is illustrated in Figure 3, the host-derived
VSMCs in these lesions also were predominantly, if not all,
derived from a non-BM source. In addition, Hu et al67 found
that after vein grafting in LacZ-transgenic normal and BMchimeric mice, approximately 40% of neointimal VSMCs
were recipient-derived whereas 60% remained donor derived.
Hillebrands et al
VSMCs and Stem Cells in Transplant Arteriosclerosis
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Figure 4. Development of TA: Alternative mechanism. Both neointimal ECs and VSMCs originate predominantly from recirculating recipient– derived precursor cells. BM does not contribute
significantly to this process.
No BM-derived neointimal VSMCs were found in this model,
thereby excluding BM as a primary source of these cells.
However, using combinations of sex-mismatched transgenic
BM-chimeric mice, Sata et al36 and Saiura et al38 showed that
in their mouse model of graft vasculopathy the BM gives rise
to most (⬇82%) neointimal cells. However, from their data it
cannot be deduced what proportion of those cells was actually
SMA-positive VSMCs. Taken together, these data indicate
that although the BM can contribute to the population of
VSMCs found in neointimal lesions, other non-BM resources
also provide precursor cells. Because most of these studies
used irradiation to create BM-chimeric animals, such hostnon-BM– derived VSMC precursors must be radioresistant.37
Obviously, ingrowth of VSMCs into the neointimal lesions from
the host side of the anastomosis cannot be excluded as a source of
neointimal VSMCs.68 However, by analyzing longitudinal sections
from the anastomosis, including both host and donor tissue,
Shimizu et al37 found no indications for ingrowth of adjacent host
medial VSMCs. Consequently a blood-borne origin of VSMC
precursors seems most likely (Figure 4). Recent data suggest that
the human peripheral blood contains a population of circulating
VSMC precursor cells. Simper and colleagues69 showed that in
vitro culture of human mononuclear blood cells in the presence of
platelet-derived growth factor–enriched medium resulted in the
generation of smooth muscle outgrowth cells. These smooth muscle
outgrowth cells displayed a variety of phenotypic characteristics of
VSMCs (eg, SMA, myosin, calponin). Whether such precursors
adhere to the luminal side of the vessel or migrate from the
adventitia through vasa vasorum toward the NI remains unknown.
Shimizu et al37 argued that sheer forces on the luminal side might
prevent adhesion of cells. At the start of NI development, however,
the first SMA-positive cells are found in a scattered pattern on the
luminal side, suggesting direct entry from the lumen.70 Also, the
observation by Sasaki et al,71 who studied human postmortem vein
graft material from patients after coronary bypass grafting, that the
process of NI formation started with the loss of ECs followed by the
385
appearance of SMA-negative spindle-shaped cells at sites of injury
suggests a blood-borne origin of these cells.
Circulating VSMC precursors might also originate from
other sources. Bucala et al72 have described a population of
non-BM– derived fibroblast-like cells in the peripheral blood
(so-called fibrocytes) that are specifically recruited from the
blood to wounded areas. Also, transdifferentiation of ECs
into VSMCs has been suggested.73 de Ruiter et al74 showed
that embryonic ECs can differentiate into SMA expressing
cells in vivo and in vitro. Whether this is a unique property of
embryonic ECs or is also shared by adult ECs remains to be
proven. SMCs themselves should also be mentioned in this
respect. Surprisingly, VSMCs (and fibroblasts) but not ECs
were able to adhere to mechanically de-endothelialized rat
aortas.75 SMCs from various locations in the vessel wall
display extensive heterogeneity.73,76 –78 Noncontractile intimal (epithelioid) SMCs with morphological resemblance to
ECs, lacking most VSMC-associated proteins, can transform
into media-like VSMCs and generate capillary tubes in vitro
consisting of ECs and VSMCs.79
One should furthermore realize that VSMCs at different
locations may have different embryonic origins.73 VSMCs in the
coronary arteries, for instance, are derived from the epicardial
lining and therefore of mesodermal origin, whereas VSMCs of
the aortic arch are of neuroectodermal origin. Furthermore,
VSMCs in the descending aorta, tissue extensively used for the
study of TA, are considered to originate predominantly from the
local mesenchyme. This could well mean that the origin of
VSMC precursors in TA also depends on the tissue studied.
VSMC Progenitor Plasticity
Given the growing awareness of plasticity of stem cells,56 –59 it
appears that neointimal VSMC precursors display similar characteristics and actually may even reside among them. We
hypothesize therefore that VSMC precursors are not a single
entity but can be recruited from a variety of resources depending
on the severity and duration of vessel damage and the critical
need for vessel repair (Table 2). In the case of limited superficial
vessel damage with a remaining vascular structure, medial/
intimal VSMCs themselves will probably provide sufficient
repair potential. More severe vascular damage, including medial
VSMC damage, over a limited period of time might signal
ingrowth of VSMCs from adjacent vessels. Severe but timerestricted vessel damage, including full disrupture of medial
VSMC layers, will lead to recruitment from non-BM sources,
whereas similar damage over a prolonged period of time will
probably need additional VSMC precursor recruitment from the
BM. Indeed, Han et al65 reported recently that BM-derived
SMA-positive VSMCs in damage-induced neointimal lesions
were only found after severe vascular damage, not in arteries
with minimal damage. Tissue damage also proved to be the
major determinant required for the transdifferentiation of primitive BM cells into cells involved in neo-angiogenesis (eg, ECs
and VSMCs) in regenerating infarcted myocardium.61,62,80 In
clinical transplantation, probably the entire spectrum of VSMC
precursor derivation can be expected to occur, perhaps even in
one and the same patient.
386
Arterioscler Thromb Vasc Biol.
TABLE 2.
March 2003
Plasticity of VSMC Replacement in TA
Damage
Little damage* over limited period
More severe damage† over limited period
Severe damage‡ over limited period
Source of VSMC
TA Development
Medial/intimal VSMC
⫾TA
Ingrowth of adjoining vessels (anastomosis?)
TA
Recruitment from non-BM sources (vasculature?)
TA
Recruitment from BM
TA
Severe damage‡ over prolonged period
*Limited medial-VSMC damage.
†Severe medial-VSMC damage.
‡Full medial-VSMC disrupture.
Acknowledgments
This work was supported by a grant of the Groningen University
Hospital. J.L.H. was supported by grants of the Dutch Diabetes
Foundation (DFN99.028 and DFN2001.05.003) and the Ter Meulen
Fund, Royal Netherlands Academy of Arts and Sciences
(TMF/DA/6419).
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Origin of Vascular Smooth Muscle Cells and the Role of Circulating Stem Cells in
Transplant Arteriosclerosis
Jan-Luuk Hillebrands, Flip A. Klatter and Jan Rozing
Arterioscler Thromb Vasc Biol. 2003;23:380-387; originally published online January 30, 2003;
doi: 10.1161/01.ATV.0000059337.60393.64
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