© 1999 American Heart Association, Inc.
Vascular Biology |
Smooth Muscle-Specific SM22 Protein Is Expressed in the Adventitial Cells of Balloon-Injured Rabbit Carotid Artery
From the Departments of Experimental and Clinical Medicine (E.F., M.P., P.P.) and Biomedical Sciences (A.C., C.M., F.S., L.Z., R.F., S.S.), University of Padua, and the CNR Unit for Muscle Biology and Physiopathology (S.S.), Padua, Italy.
Correspondence to Angela Chiavegato, PhD, Department of Biomedical Sciences, University of Padua, Viale G Colombo, 3, I-35121 Padua, Italy. E-mail smggroup{at}civ.bio.unipd.it
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Abstract |
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Abstract—During the "response-to-injury" process after a mechanical insult to the porcine coronary arteries, the adventitial cells acquire the structural characteristics of myofibroblasts before being incorporated into smooth muscle (SM) layer. We assessed whether the SM-specific SM22 protein can be used as a tracer of adventitial cell-myofibroblast differentiation in the mild balloon injury of rabbit carotid artery. To achieve this goal, we used 2 monoclonal anti-SM22 antibodies (E-11 and 1-B8) and a molecular probe for the SM22
mRNA isoform in
immunocytochemical and in situ hybridization experiments. The
differentiation profile and the migratory and proliferative ability of
activated adventitial cells were evaluated by a panel of
antibodies to some SM and nonmuscle antigens and pulse- and
end-labeling with bromo-deoxyuridine, respectively. In adventitial
cells, SM22 antigenicity and SM22 mRNA were detectable at days 2 and
4 and, to a lesser extent, at days 7 and 21 after injury, particularly
near the adventitia-media interface and mostly colocalizing with
bromo-deoxyuridine–positive cells. The pulse-labeling experiments
showed that the large majority of these cells penetrated the outermost
layer of the tunica media without migrating to the
subendothelial region. The phenotypic features of
activated migrating and nonmigrating adventitial cells
resembled those of vimentin-actin myofibroblast subtype and fetal-type
SM cells. These findings indicate that a direct exposure of adventitia
to the lumen is not required for phenotypic changes and
proliferation/migration of these cells. After comparison of the SM22
expression in arterial vessels during early stages of
development, we hypothesize that in the injured carotid artery the
mural incorporation of adventitial cells and the spatiotemporal
activation of SM22 expression are reminiscent of the vascular
morphogenetic process and suggest the existence of a stem cell-like
reservoir in adventitia. The early adventitial upregulation of SM22
expression in the injured vessel might be related to a multistep
transition process in which nonmuscle cells are converted to
myofibroblasts and, possibly, to SM cells.
Key Words: smooth muscle cell • adventitia • differentiation • endothelial injury • SM22
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Introduction |
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Abasic aspect of the pathogenetic mechanism of atherosclerosis is the process of proliferation and migration of arterial smooth muscle cells (SMC) from the media to the subendothelial region.1 2 3 A similar phenomenon is observed in the endothelial-injured vessel, in which accumulating neointimal SMC derive essentially from the underlying medial tissue.2 4 The adventitial layer has so far been considered as just a site of perivascular inflammation and tissue healing, which might be involved in wall remodeling, but without any contributing role to the atherosclerotic lesion or neointimal formation.1 2 5
Recent works have, however, suggested that the adventitia may play an important role in atherosclerosis or neointima formation after endothelial injury. In fact, microischemia of vasa vasorum6 7 8 and activation of resident9 10 11 12 or blood-derived13 nonvascular cells have been implicated in the arterial wall lesion formation. According to the former proposed mechanism, in thick-walled large vessels containing thrombotic vasa vasorum, hypoxia produces foci of necrosis in the medial smooth muscle (SM) tissue, which in turn activate an SMC regeneration process. As for the latter proposed mechanism, the adventitia of injured coronary artery displays a cellular transition from fibroblasts to myofibroblasts,12 ie, a cell phenotype with hybrid or intermediate structural and functional properties between fibroblast and the SMC.14 15 There is now convincing evidence that these adventitial myofibroblasts possess directional (centripetal) migratory ability and can be, to a certain extent, incorporated in the tunica media and, possibly, in the neointima.16
If adventitial myofibroblasts derive from locally recruited fibroblasts or fibroblast-like cells,12 one might consider the possibility that the cellular conversion process described above might just be an expression of the differentiable plasticity of fibroblasts as occurs in villous placental stroma,17 in some experimental models,18 19 20 and in some human pathological settings.21 Alternatively, such cell phenotypic transition might represent a reactivation of the mesenchymal cell pathway, which during development is involved in the morphogenesis of arterial wall.3 In this latter circumstance, it is plausible that a sort of embryonic remnant may persist in the adult at the level of adventitia, ie, in a region where during vasculogenesis the precursors of differentiating SMC (undifferentiated mesenchymal cells surrounding the endothelial tube) are thought to be localized.3
In light of these hypotheses, it is important to ascertain in more
detail the differentiable potential of activated adventitial
cells in a model of endothelial injury of carotid
artery. We have undertaken this study, taking into account the SM22
protein22 23 as a principal marker, to study the
phenotypic changes in the adventitial cells. This calponin-related
protein is composed of 4 electrophoretically distinguishable isoforms
(, ß, 
, and 
),22 24 25 being the most
thoroughly investigated.26 27 28 The SM22 expression in
adult is restricted to SM tissues,22 23 24 whereas in the
early stages of development SM22 is also present in skeletal and
cardiac muscle.29 In muscle tissues other than SM tissue,
SM22 is switched off during birth or hatching.23 28 29
The distribution patterns of SM22 found at the protein level in normal
and injured rabbit carotid artery at various times of injury (2, 4, 7,
and 21 days) were compared with that found in in situ hybridization
(ISH) experiments for SM22 mRNA. Immunophenotyping of adventitial
cells was accomplished by a panel of antibodies specific for SMC
lineage-related (SM-type -actin and calponin), SMC-specific (SM-type
myosin heavy chain [MyHC] and h-caldesmon) proteins,
cytoskeletal (vimentin, desmin, and nonmuscle [NM]-MyHC) and
extracellular matrix (ECM; EIIIA-fibronectin) markers. Experiments of
pulse- and end-labeling of proliferating SMC with bromo-deoxyuridine
(BrDU) were also designed to evaluate whether phenotypic modifications
were related to the migratory and proliferating ability of adventitial
cells and SMC.
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Methods |
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Animals
Aortic specimens to be used in 2-dimensional gel electrophoresis (2D-EF) and carotid samples to be processed in immunocytochemistry experiments were taken from 3-month-old male New Zealand White rabbits obtained from C & C Farm (Padna, Italy) (weighing about 3 kg), and day 12 rabbit fetuses. Forty-six adult rabbits were used in the endothelial injury experiments: 16 underwent surgery and 24 were subjected to carotid lesion and BrdU incorporation (pulse or end labeling). Three untouched animals were used as controls. Balloon-injured rabbits were sacrificed at 2, 4, 7, and 21 days after surgery (see Figure 1
). For each time
point animal groups comprised 4 (operated) or 3 (operated+pulse
labeling, or operated+end labeling) rabbits. Rabbits were housed in the
Department of Biomedical Sciences and quarantined for 7 days before
use. Maintenance of animals was done in accordance with the
recommendations stated in "Principles of Animal Care" (NIH
publication No. 85 to 23, revised 1985) and the "Guidelines of the
Animal Care Advisory Committee" of the Italian Ministry of Public
Health. Euthanasia of all rabbits was performed by sodium
pentothal injection.
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Endothelial Lesion
Animals were anesthetized with sodium pentothal (30
mg/kg; Abbott Laboratories) and urethane (70 mg/kg), administrated via
a marginal ear vein. A 2F Fogarty embolectomy catheter (Baxter
Healthcare Co) was introduced through an aseptic neck incision produced
in the facial branch of the external left carotid artery and positioned
approximately at the origin of the common carotid. An acute balloon
injury was performed by inflating the balloon with 0.2 mL saline
solution and then gently pulling it back along the entire length of the
common carotid with constant rotation. The procedure was repeated 3
times. The catheter was then removed, the artery branch ligated, and
the surgical wound closed. The animals were allowed to recover under
observation before being placed in their cages. Evans blue dye
injection 2 hours after surgery confirmed the near
homogeneous removal of endothelium from
injured carotid. Histological examination of this area
revealed a marked structural alteration of SMC in the presence of a
substantially intact wall organization. Because the lesion did not
apparently involve disruption of the medial integrity (ie, the elastic
membranes) and contact of the blood with the adventitia, we define the
impact of the lesion on the carotid wall as mild injury.
After sacrifice, the left common carotid artery and the contralateral vessel were excised with great care to avoid any damage to the adventitial layer. The vessel segments were then perfused with OCT (Tissue Tek, Miles Inc) and inserted into the lumen of the thoracic aorta from the same animal. The mounted block was immersed in liquid nitrogen and stored at -80°C. Contralateral carotid artery was used as a control.
Two-Dimensional Gel Electrophoresis
2D-EF for basic proteins at nonequilibrium pH condition was
performed as outlined by O'Farrell30 and Gimona et
al25 with the following modifications: ampholytes (Serva)
covered pH ranges 5 to 8 (2.5%), 4 to 6 (1.6%), 3 to 5 (1.6%), 7 to
9 (1.6%), and 2 to 11 (0.8%). Isoelectric focusing was performed
according to the following protocol: 400 V (7 minutes), 500 V (7
minutes), and 750 V (75 minutes), without a preliminary run. The
second-dimension electrophoresis was done in a 15% SDS-slab gel.
Western blotting of electrophoresed protein bands was done using parallel gels and following the standard conditions used in our laboratory31 32 with some modifications. Transfer of proteins from the 2D gels to the nitrocellulose paper was performed for 90 minutes at 300 mA. Free binding sites were saturated in 5% fat-deprived milk, and E-11 or 1-B8 primary antibody was applied to the paper for 30 minutes at 37°C. The secondary antibody was an IgG anti-mouse IgG coupled with horseradish peroxidase (Dako) applied to the paper at the same conditions as the primary antibody. Bound primary-secondary antibody complex was revealed by chemiluminescence using the Amersham ECL kit.
Pulse- and End-Labeling
Injured animals treated with 30 mg/kg of BrdU, dissolved in PBS,
pH 7.2, were injected IP 12 and 24 hours after surgery (pulse labeling;
see Figure 1
) and euthanized at 2, 4, 7, and 21 days after
injury. Another group of injured rabbits injected with the same dose of
BrdU 12 and 24 hours before killing (end labeling; see Figure 1)
were euthanized at 2, 4, 7, and 21 days after injury. Three animals not
operated on were also treated with BrdU and used as control group.
Antibodies
A panel of monoclonal antibodies was used to characterize
immunocytochemically the adventitial cell phenotype at various
times from injury induction. The specificity of the antibodies is
reported in Table 1.
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Immunocytochemistry
Primary antibodies were applied to freshly cut fixed or unfixed
cryopreserved sections as previously described.31 32
Cryopreserved sections were taken from the middle part (about 1 cm) of
the common carotid artery. For immunophenotyping of adventitial cells,
15 carotid cryopreserved sections from each control and injured animal
were used. For cell proliferation/migration study, 10 carotid
cryopreserved sections from each time point from control,
injured+pulse-labeled, or injured+end-labeled rabbits were
analyzed. For E-11 and 1-B8, anti–h-caldesmon and
anti–endothelin-1 antibodies, cryopreserved sections were fixed in
3.7% formaldehyde in PBS, pH 7.2, for 5 minutes.
Anti–phosphoglucomutase-related protein (PGM) antibody and
anti-laminin were applied to methanol- or acetone-fixed cryopreserved
sections, respectively. In these procedures, the secondary antibody was
the IgG anti-mouse IgG coupled with horseradish peroxidase. Bound IgG
were revealed by incubation in aminoethylcarbazole solution.
Counterstaining was performed with Harris' hematoxylin. Controls were
made by omitting the primary antibody and using nonimmune IgG followed
by the secondary antibody. The double
immunofluorescence assays were performed using a
monoclonal anti–SM-type -actin (Sigma) directly coupled with
fluorescein isothiocyanate and the anti-BrdU antibody
indirectly revealed with the anti-mouse IgG conjugated with rhodamine
isothiocyanate.32
BrdU-incorporating nuclei in normal and injured vascular tissue from pulse and end labeling were detected using a specific anti-BrdU antibody (Dako) according to the procedure reported by Faggian et al.19 Negative and positive BrdU-incorporating tissues (myocardium and small intestine, respectively) from the same animals used for BrdU labeling of injured vessels were used as controls.
Variability of immunophenotyping and BrdU-incorporation patterns from section to section were approximately on the order of ±10% of positive cells. The intensity of immunostaining in the carotid cryopreserved sections, especially at early stages of the response-to-injury process, was generally limited and apparently depended on the existence and size of the thrombus over the injured region (Chiavegato A, Franch R, Sartore S, in preparation, 1998).
In Situ Hybridization
The pBluScriptSKII+ vector containing a
275-bp fragment of rat SM22-cDNA29 (a generous gift of
Dr Joseph M. Miano, Cardiovascular Research Center,
Department of Physiology, Medical College of Wisconsin) was linearized
with the appropriate restriction enzymes. Sense and antisense RNA
probes labeled with 35S-UTP were transcribed with
T3 and T7 polymerases according to the manufacturer's instructions
(GibcoBRL). ISH was carried out on 10-µm cryopreserved sections
collected onto 3-aminopropyl-triethoxysilane–treated slides. First ISH
cryopreserved sections were fixed for 30 minutes with 4%
p-formaldehyde in PBS, pH 7.2, and then processed following
the protocol of Sassoon et al34 and DeNardi et
al35 with some modifications. After rinsing in PBS, the
cryopreserved sections were subjected to 4 minutes of digestion in 10
µg/mL proteinase K at room temperature, followed by 0.1 mol/L
triethanolamine-acetic anhydride acetylation and
dehydration. For hybridization, riboprobes were heated for 3 minutes at
90°C in 50% formamide, 0.3 mol/L NaCl, 0.3 mol/L Tris-HCl (pH 7.4),
5 mmol/L EDTA (pH 8.0), 10 mmol/L
NaH2PO4 (pH 8.0), 10%
dextran sulfate, 1xDenhardt's solution, 0.5 mg/mL yeast tRNA, and
10 mmol/L DTT, and placed on ice.34 Approximately 60
µL of probe was applied to each slide, a coverslip was applied, and
probes were hybridized in a humidified chamber at 50°C for at least
16 hours. The remainder of the ISH procedure was identical to that of
DeNardi et al.35
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Results |
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Specificity of Antibodies
Isoform specificity of the 2 anti-SM22 antibodies in the vascular SM tissue is shown in Figure 2
. Two
electrophoretically distinct spots of 22-kDa, corresponding to the
and ß isoforms of SM22, can be seen in the gel (Figure 2A).
1-B8 and E-11 antibodies react strongly with the isoform, whereas
E-11 is weakly positive with the ß isoform (Figure 2B). No
cross-reactivity of the 2 antibodies with the calponin is detectable
(Figure 2B and 2C).
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Immunophenotypic Characterization of Adventitial Cells in Normal
and Injured Carotid Artery
Figure 3 shows the
immunocytochemical reactivity of adventitial cells and the overlying
SMC in normal and injured carotid artery at various times from
endothelial lesion with anti-SM22 antibodies. Because
the SM22 immunostaining of the vascular tissues with
the 2 antibodies is similar, only data pertaining to E-11 will be
presented here.
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In the adventitia from normal wall, there is almost no reactivity with
resident cells (Figure 3A) except for rare vasa vasorum (not
shown), whereas a marked staining is visible with the medial SMC. In
uninjured carotid artery, there is no histological
evidence for a tunica intima or intimal cushions. In injured vessel,
SM22-positive adventitial cells are clearly identified at all
postinjury times examined: from day 2 (Figure 3B) to day 21
(Figure 3E). However, at day 2 and, to a lesser extent, at day 4
the SM22 appears to be localized near the adventitia-media interface,
whereas at later times SM22-positive cells are dispersed throughout the
adventitial layer. Small areas of neovascularization are generally
localized in the outer region of adventitia where the vasa vasorum are
found.36 In the media, there is a downregulation of SM22
cellular content, particularly around day 4 after injury (Figure 3C), which is reversed at later stages (Figure 3D and 3E), although SMC heterogeneity can persist in some
regions (see for example Figure 4G).
Neointimal cells are almost negative for SM22 at day 7
(Figure 3D), but become markedly positive at day 21 (Figure 3D) with some slight differences in the intensity level from
region to region.
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The presence of SM22-containing adventitial cells in injured carotid
artery was also confirmed at the mRNA level. Figure 4C shows the
result of ISH experiments performed on day 2 balloon-injured vessel in
comparison with the uninjured carotid (Figure 4B) and the
corresponding sense probe (Figure 4A). In concordance with data
obtained at the protein level (Figure 3B), small foci of SM22
mRNA are identified in the adventitial region proximal to the external
elastic lamina. Similar results were also found with day 4 injured
carotid artery (not shown). In contrast, some differences exist between
expression of protein and mRNA levels in the media. As might also be
inferred by comparing Figure 3C with Figure 4C, there is
in this tissue an apparent discrepancy between a relatively decreased
SM22 expression at day 4 and an increased level of SM22 mRNA at day
2. To study this problem in detail, cryopreserved sections from day 4
(Figure 4D and 4E) and day 21 (Figure 4F and 4G) injured
carotid arteries, which appeared heterogeneously stained
with the anti-SM22 antibody, were taken and compared with parallel
cryopreserved sections prepared according to the ISH protocol. Areas
highly reactive for both SM22 protein and SM22 mRNA coexist along
with areas in which the protein is more or less detectable in
comparison with SM22 mRNA. The fact that the SM22 expression at the
protein level is low and the SM22 mRNA is high might indicate that a
regulatory mechanism controls the SM22 expression at the mRNA level.
The opposite relationship between the relative protein and mRNA levels
might be attributable to the upregulation of non-SM22 isoform
expression recognized by the anti-SM22 antibodies used in this
study.
Figures 5 and 6 and Table 2 show the results of the
immunophenotyping experiments performed to characterize the normal and
activated adventitial cells. In normal carotid artery the
resident adventitial cells (not including nerves, vasa vasorum, and
lymphatics) can be identified as fibroblasts,14 15 ie,
vimentin-9 10 11 and NM-MyHC-33 positive cells
(Table 2). On vessel damage, part of the cells near the
adventitia-media interface subsequently express a cytoskeletal and
cytocontractile marker repertoire typical of VA (vimentin-actin)
-myofibroblasts, ie, reactivity for vimentin, actin, and
NM-MyHC,14 15 18 33 along with SM22 (Figure 5C).
Small foci or isolated cells in this region are labeled for SM myosin
(Figure 5E), the SM-related protein PGM (Figure 5F),
fibronectin (Figure 6G), and, rarely, calponin (Figure 5B). Other SM-specific markers, such as caldesmon (Figure 5D) and desmin (Figure 6B) are not reactive. The
outermost layer of the adventitia is composed of vimentin- (Figure 6A) and NM MyHC- (Figure 6C and 6D) positive fibroblasts.
Endothelin-1 expression also appears (Figure 6E). Inflammatory
cells identified by RAM11 (Figure 6H) are barely visible at 2
days after injury. Cells positive for the various antibodies do not
morphologically resemble cells of nerve, vasa vasorum ,or lymphatics.
Taken together, these results are compatible with the presence of a
2-layer organization in adventitia of day 4 injured carotid artery: the
outer region is composed of fibroblasts, whereas the inner region is
composed of SM22-containing myofibroblasts and SMC.
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The medial SMC in the injured artery display predominantly the
phenotypic features of fetal SMC, namely positive for SM myosin,
NM-MyHCpla1/2,33 reduced staining of
calponin (Figure 5B), SM22 (Figure 5C), caldesmon (Figure 5D), desmin (Figure 6B), and laminin (Figure 6F),
and increased staining for fibronectin (Figure 6G). Endothelin-1
expression is also evident (Figure 6E). Interestingly, in some
medial regions facing the lumen there are NM cells, positive for
vimentin (Figure 6A) and NM-MyHC (Figure 6C and 6D),
which can be identified only in part as inflammatory RAM11-reactive
cells (Figure 6H) or lymphocytes (not shown).
Proliferation/Migration of Adventitial Cells
Pulse- and end-BrdU labeling were used in injured carotid artery.
The former procedure allows for monitoring the waves of migratory cells
that might be induced at various times from injury in the wall
compartments assuming that cells incorporating this thymidine analog
become almost completely arrested in the S-phase of the cell cycle
without being blocked in their migratory capability.37 38 39
Generally, the intensity of BrdU immunostaining is
quite high and homogeneous, suggesting that a diluting
effect consequent to BrdU incorporation and associated with cell
proliferation is scarce. We are aware of the possibility, however, that
BrdU-positive cells may maintain a relatively high immunocytochemical
signal in the daughter cells after the first division. The second
assumption is that the end labeling furnishes information about the
localization of proliferating cells during the 24-hour window preceding
euthanasia at 2, 4, 7, and 21 days after injury.
In uninjured vessel, only rare BrdU-positive cells are present in
the media and adventitia (not shown), whereas numerous
BrdU-incorporating cells are evident at the different postinjury times
examined (see Figure 7). In the pulse
labeling, the large majority of reactive cells are confined to the
adventitia layer (see Figure 7A through 7G), although a limited
number of cells, especially at 2 and 4 days after injury, can also be
seen in the media just near the external elastic lamina (Figure 7A and 7C). Interestingly, numerous adventitial BrdU-positive
cells appear to be localized proximal to the external elastic lamina,
remarkably at the early stages after injury.
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The end-labeling procedure gives rise to a more complicated picture
than pulse labeling. At 2 days after injury (Figure 7B), the
distribution of BrdU-positive cells is quite similar to that of the
corresponding pulse-labeled artery (Figure 7A), inasmuch as the
times of BrdU injections in the 2 protocols are close to each other
(see Figure 1). Hence, the majority of BrdU-positive mural cells
belong to the adventitial compartment (Figure 7B). At day 4
(Figure 7D), only very few BrdU-incorporating cells are visible
in the adventitia, being mostly localized in the
subendothelial region of the media. At the subsequent
times, cells reactive with BrdU decline dramatically and at 21 days
after injury (Figure 7H), no positive ones can be identified in
the carotid wall. It is worth noting that a neointima is
formed in the carotid artery from both types of BrdU protocols, but in
contrast with the end-labeling procedure, in pulse labeling almost no
BrdU-positive cells are seen in this tissue and the
neointimal thickening is thinner (Figure 7E).
Our data are compatible with the idea that 2 temporally distinct waves of cell migration take place in the injured carotid artery wall: a first adventitial wave followed by a second medial SMC wave. In our experimental conditions, it seems that adventitial cells are short-range (directional) migratory cells and do not contribute substantially to neointima formation.
We have also sought to characterize the phenotypic profile of
activated cells localized at both sides of the external elastic
lamina. Figure 8 shows the result of
double immunofluorescence experiments performed on
carotid artery from a day 2 BrdU-pulse- labeled injured animal. The
large majority of cells are stained for SM-type -actin and, thus,
from the results shown in Figures 5 and 6 and Table 2, they can be identified as VA-myofibroblasts.
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The fact that small groups of adventitial cells are reactive for some
SM-specific markers (see Figure 5; in particular, SM myosin
being the only reliable marker of SMC differentiation40 )
poses the problem of evaluating their differentiation level using the
combination of anti-SM myosin and anti–NM-MyHC
antibodies.33 Secondly, it must also be verified whether
SM22- and SM myosin-expressing cells have the ability to penetrate the
adventitial-medial barrier, ie, if they possess a migratory ability.
Figure 9 shows that the small foci of
adventitial cells positive for SM myosin (Figure 9A) found at 2
days after injury are also labeled for
NM-MyHCpla1/2 (Figure 9B and 9C), thus
indicating that these are fetal SMC.33 Secondly, in no
circumstance are medial SMC proximal to the external elastic lamina
negative for SM myosin and positive for
NM-MyHCpla1/2 (the expected pattern for a true NM
cell), indicating that fibroblasts as such do not enter into media. In
contrast, postnatal (SM myosin-positive,
NM-MyHCpla2-positive) and fetal (SM
myosin-positive, NM-MyHCpla1/2-positive) SMC are
visible at the luminal side of the media, as expected from the inferred
data of end labeling (ie, the second medial SMC wave described above).
Serial cross-section immunostaining with anti-SM myosin
and anti-SM22 antibodies show that BrdU-positive cells appearing at
both sides of the external elastic lamina (Figure 9F and 9G)
also display an SM myosin (Figure 9D) or SM22 (Figure 9E)
content, suggesting that fetal SMC and SM22-containing
VA-myofibroblasts are capable of penetrating the adventitial-medial
barrier.
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Some Immunophenotypic Properties of Developing Aortic Wall
Because the directional migration of adventitial cells toward the
lumen in the endothelial-injured model in adult rabbit
might be reminiscent of the first stages of vascular morphogenesis (see
Discussion), we have also looked at spatial distribution of SM22
antigenicity in developing aorta (12-day-old fetus; Figure 10). At this phase of vasculogenesis
the wall organization is poor with no evidence for media-adventitia
demarcation or an organized medial layer with the characteristic
elastic laminae. It is generally accepted that in this period newly
incorporated (in the developing vessel) SM -actin-positive cells
come from locally recruited mesoderm-derived mesenchymal precursors
surrounding the hollowed endothelial
tube.3 The parietal cells close to
endothelium that we have examined are not yet composed
of fully differentiated SMC as witnessed by the lack of reactivity with
anti-SM myosin antibody (Figure 10C). By contrast, SM -actin
expression is visible both close to the lumen (as a relatively compact
immunostaining) and in the surrounding cells (in a
dispersed manner; Figure 10A). SM22 immunoreactivity is greatly
expressed, in part overlapping that of actin but also encompassing a
thicker wall layer (Figure 10B).
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Discussion |
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In response to mild injury to rabbit carotid artery 2 important phenomena take place: (1) the adventitial cell activation accompanied by phenotypic changes related to the acquisition of SMC-related properties and (2) migration of adventitial myofibroblasts/SMC and medial SMC to the media and neointima, respectively.
Phenotypic Changes in the Adventitia
The results that support the first point are obtained by studying
the expression of SM22, an SM-specific protein in adult
avian23 25 and mammalian26 27 28 29 species, in
comparison with a number of SM- and NM-specific antigens that have
allowed the identification of 2 major cell types: the myofibroblasts
and SMC. Myofibroblasts, which appeared during the wound repair process
consequent to the application of a mechanical insult to the vascular
wall, are characterized by the upregulation in the expression of
SM-type -actin in the preexisting vimentin- and NM myosin-containing
adventitial fibroblasts. This new cell phenotype can be
classified as VA-myofibroblast on the basis of the nomenclature
proposed by Gabbiani and coworkers.14 15
Our immunophenotyping procedure has revealed a peculiar characteristic
of adventitial myofibroblasts: the expression of the SM
lineage-specific marker SM22. It is highly probable that at least the
isoform of SM22 is present in the activated adventitial
cells, though we cannot exclude that upregulation of the ß, , or
variants22 24 25 may also occur during the
arterial wall response-to-injury process. Because the
expression of the calponin-related SM2223 or
SM2228 29 is generally confined to SM tissues, the
presence of this protein might be quite unexpected. There are, however,
some data that suggest that SM22 is also expressed in myofibroblasts
from other experimental settings or in NM cell systems. In fact,
serosal myofibroblasts from rabbit urinary bladder subjected to partial
outflow obstruction19 or transforming growth factor-ß1
infusion41 contain SM22-expressing VA-myofibroblasts. SM22
is also expressed in vitro in rat embryo42 and human
senescent43 fibroblasts, in the former as an actin gelling
protein, transgelin.42 In addition, SM22 is present in
cultured IMR-90 human fetal lung cells showing the structural
characteristics of myofibroblasts.44 Altogether, the
differentiation profile of activated adventitial cells
resembles that of some SMC-like lines in which SM22, actin, and NM
myosin are coexpressed.45 46
The fact that in our model of endothelial injury some
adventitial cells in the region proximal to the external elastic lamina
display an SM myosin content (a marker of fully differentiated
SMC40 ) points to the existence of a differentiation
pathway in which myofibroblasts can be converted into SMC. On the basis
of our SMC immunophenotypic criteria,33 47 these SMC can
be identified as fetal SMC (Figure 9A through 9C), ie, showing
coexpression of SM and NM-MyHC-Apla1/2 isoforms
as well as the fetal-fibronectin isoform.48 49 Thus, SM22
expression in adventitial cells can be considered as an indicator of
the spatiotemporal cellular transition that brings about the
myofibroblast to SMC transformation. This implies that the cellular
conversion of myofibroblasts into SMC may occur independently from the
migration into the media, possibly indicating that injured adventitia
contains locally released factors that can allow this process to take
place.
The presence of adventitial-derived myofibroblasts/SMC near the
external elastic lamina and the tendency for the spatial-limited
migration toward the lumen (see below) are reminiscent of the
incorporation-differentiation process that some arterial
vessels undergo during vascular morphogenesis.3 In fact,
undifferentiated SMC precursors migrating from the mesodermal region
surrounding the endothelial tube are incorporated into
the developing vessel wall.3 Looking at Figure 10, showing the dorsal aorta from a day 12 rabbit fetus, it seems that a
continuum of SM22-positive cells exists between the already established
actin-containing aortic wall SMC and the surrounding tissue in which a
real adventitia has yet to be formed. Although other hypotheses are
possible, we put forward the idea that adult adventitia may contain a
sort of a stem cell-like population that can be activated on
demand, possibly differentiating according to an adventitial-medial
axis resembling a vasculogenetic pathway.
Migration of Activated Vascular Cells
The results of pulse- and end-BrdU labeling experiments are
compatible with the existence of 2 distinct migrating/proliferating
cell responses in injured carotid artery wall, namely, adventitial cell
proliferation-incorporation into the outer medial layer, and the
proliferation-migration of medial/neointimal SMC.
Pulse-labeling studies have shown that the large majority of
adventitial cells and, to a lesser extent, cells in the outer layer of
the tunica media incorporate BrdU soon after lesion. The time-course
analysis of distribution of BrdU-incorporating cells also
reveals that adventitial cell migration to the medial layer peaks
around 2 days after injury and declines thereafter, possibly because of
the decreased density of medial BrdU-positive cells (caused by
increased total medial SMC2 ).
Because there are almost no labeled cells in the neointima, according to the pulse-labeling procedure, it might be concluded that in our experimental model BrdU-positive cells accumulated in this tissue come mostly from the underlying medial SMC. In fact, the results of the application of end-labeling protocol at 4 and 7 days after injury indicate that the innermost medial region and neointima contain proliferating cells. Taken together our data suggest that 2 waves of migration/proliferation exist at the 2 interfaces of tunica media, ie, at the level of external and internal elastic lamina.
Some caution must, however, be taken about the conclusions drawn from the use of BrdU as a tracer of cell migration from 1 vascular compartment to another, particularly regarding the absence of BrdU in the neointima described above. Using the BrdU approach to this problem, it cannot be ruled out that the stability of this DNA-incorporated thymidine analog is maintained during cell migration. In addition, it might be that not all the activated cells are labeled by BrdU or that repeated cell division may cause some loss of the label. Future studies dealing with this problem must take into account other procedures, for example the use of a fluorescent dye applied to adventitial cells before injury to trace the fate of newly migrating cells.
BrdU Incorporation and Cell Differentiation
The 2 waves of proliferating/migrating cells in the injured
carotid wall are accompanied by distinct time-dependent differentiable
events: (1) differentiation of adventitial cells into myofibroblasts
and then, in part, into SMC, and (2) dedifferentiation of
adult-type into fetal-type SMC (or expansion of preexisting postnatal
SMC).33 It is worth noting that in our model, in
concordance with Shi et al,11 adventitial NM cells are not
capable of migrating to the media as occurs with myofibroblasts or
fetal SMC. It could be that SM22 expression in NM cells might confer
some migratory advantage as inferred from this peculiar propensity that
the SM22-positive NM cells (named type 250 ) from the
adventitial surface of canine carotid artery show when grown in
vitro.51 It would be of interest to investigate a possible
correlation between the severity of injury and the level of
upregulation of SM22 expression in adventitia.
Our data support the concept that competence for SMC migration from the
media to the subendothelial space requires the
dismantling of the differentiable apparatus of the cells,
ie, an upregulation in the expression of NM cell markers (NM myosins,
fibronectin, and vimentin) and downregulation of SM markers (SM myosin,
caldesmon, calponin, PGM, and SM22). The neointima is
initially poor of SM22-positive cells, but it gains a more abundant
expression of SM22 when the proliferation level is scarce or negligible
(Figure 3). Our results are in concordance with data from
Shanahan et al,52 53 who found that SM22 expression is
quite low in human atherosclerotic plaque,52 and with the
in vitro differentiation-dependent expression of this
polypeptide.53
There is still an open question represented by the presence
of NM cells in the inner medial layer that are negative for SM markers
including SM22 (Figure 3) and cannot be accounted entirely for
contaminating inflammatory cells. Further studies are needed to clarify
the exact derivation of this cell type.
Comparison With Adventitial Cell Activation in Overstretching
Injury Model
Using the overstretching model of coronary artery
lesion, Scott et al9 and Shi et al10 11 have
come to a similar conclusion about the phenotypic changes and the
migratory properties of adventitial cells. Some differences, however,
are noticeable. Contrary to the severe injury produced in the porcine
coronary artery by these authors, the adventitia-to-media cell
migration observed in our model is modest. In fact, in pulse-labeling
tests only a few BrdU-positive cells can be seen dispersed in the media
at day 4 or 7 (see Figure 7C and 7E). The neointima
produced is scarce with rare BrdU-positive cells. In adventitia, the
phenotypic changes from NM to myofibroblasts occur faster (2 days), and
we have shown that SMC differentiation can take place before
adventitial cells migrate to the media. Although in the porcine injury,
adventitial cell proliferation index peaks around 3 days after injury
and the maximal level of phenotypic changes develops around day 7 to
14, in the rabbit model the waves of proliferation and phenotypic cell
transition occur concomitantly.
To sum up, our data indicate that a direct exposure of adventitia to the lumen is not required for phenotypic changes and proliferation/migration of these cells. The difference observed in the porcine versus the rabbit model of injury might be because of the extent of lesion, the procedure to induce vessel damage, and the species used in the experiments.
Perspectives
Conclusive evidence for the fibroblast derivation and the factors
potentially accompanying or inducing the NM cell to myofibroblast (to
SMC) transition in the adventitia of injured carotid artery is lacking.
Future studies must establish in detail whether other NM cell types in
addition or alternative to fibroblasts are responsible for such
phenomenon. In particular, pericytes14 15
endothelial cells,54 55 SMC from vasa
vasorum,56 and CD34+ mononuclear
blood cells13 should also be taken into account.
Certainly, the transient perivascular inflammatory
reaction57 and ischemia of vasa
vasorum6 7 induced soon after endothelial
injury may cause the release of a number of growth
factors/cytokines, which may have a profound effect on the
subsequent activation of local resident cells or blood- and SM-derived
cells. Among the factors putatively involved,
endothelin-1,58 59 as demonstrated in this study (Figure 6E and Table 2), transforming growth
factor-ß1,60 and platelet-derived growth
factor9 seem to be good candidates. It remains to be
established whether and how the process of
activation-recruitment-incorporation of adventitial cells in the
media (and neointima) is operative during
atherogenesis.
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Acknowledgments |
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This work was supported in part by the Biomedical Association for Vascular Research of Padua, Italy. We thank Marissa Galliani-Sanavio for her excellent editorial assistance.
Received July 24, 1998; accepted October 7, 1998.
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References |
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