© 1997 American Heart Association, Inc.
Articles |
Upregulation of Connexin43 Gap Junctions Between Smooth Muscle Cells After Balloon Catheter Injury in the Rat Carotid Artery
From Imperial College School of Medicine, National Heart and Lung Institute (H-I.Y., E.D., N.J.S.), and the Thrombosis Research Institute (F.L.), London, UK.
Correspondence to Professor N.J. Severs, Cardiac Medicine, Imperial College School of Medicine, National Heart and Lung Institute, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. E-mail n.severs{at}ic.ac.uk.
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Abstract |
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Abstract Phenotypic transformation of smooth muscle cells (SMCs) to the synthetic state in vitro and in human coronary atherosclerosis is reported to be associated with upregulation of connexin43 gap junctions. To determine whether cellular interactions mediated by gap junctions participate in the phenotypic transformation of SMCs in arterial injury and disease in general and to establish the spatial and temporal pattern of any such change in relation to neointimal development, we investigated SMC connexin43 gap junction expression during vascular healing in the rat carotid artery after balloon catheter injury. Quantitative immunoconfocal microscopy was applied to localize and to quantify connexin43 gap junctions 1, 3, 9, and 14 days after injury. Parallel studies were conducted by electron microscopy (direct morphological demonstration of SMC gap junctions) and immunoconfocal microscopy (localization of altered actin expression). Synthetic-state SMCs in the neointima (first apparent from 9 days postinjury) revealed abundant expression of gap junctions, with levels of immunodetectable connexin43 threefold greater than those of medial cells. However, the first detectable changes were found in the media, before neointimal formation; at 1 to 3 days postinjury, an increase in SMC gap junction expression was apparent in the innermost (subluminal) zone, the major site from which the cells subsequently found in the neointima are recruited. We conclude that upregulation of connexin43 gap junctions is intimately linked to SMC phenotypic transition and that interactions mediated by gap junctions may be a hitherto unrecognized contributor to the cellular mechanisms underlying the vascular response to injury.
Key Words: gap junctions • connexin43 • smooth muscle cells • balloon injury • neointima
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Introduction |
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Migration and proliferation of smooth muscle cells (SMCs), and synthesis of extracellular matrix by these cells, are key events underlying atherosclerotic disease and the healing response to vascular injury following balloon angioplasty, stent implantation, and atherectomy.1–4 Although the precise pathogenetic features of these processes differ in specific detail, all involve transformation of SMCs from the differentiated contractile state to the activated synthetic state.5,6 Such phenotypic transformation is initiated and maintained by interactions between SMCs and other cells or constituents of the arterial wall and blood. Interactions involving extracellular signaling mechanisms, such as those mediated by growth factors and cytokines, have been extensively studied in this context,7,8 but more recently another form of cell-to-cell communication, that mediated by gap junctions, has also been implicated.9,10 Furthermore, other evidence suggests interactive effects between these two communication mechanisms.11–13
Gap junctions comprise clusters of transmembrane channels that act as conduits for the direct intercellular exchange of ions, secondary messengers, and small signaling molecules, a key function to tissue homeostasis and the regulation of growth, differentiation, and development.14–17 The component proteins of gap junctions, connexins, form a multigene family of conserved proteins, of which 13 members have now been reported in mammalian cells.14,18 The precise pattern of connexin expression varies according to cell type and tissue, and gap junction channels made from different connexin types have distinct functional properties.19–21 The principal connexin isoform expressed by vascular smooth muscle is connexin43. Although coexpression of this connexin with another isoform, connexin40, has been reported in the smooth muscle of some microvessels and in one SMC line,9,10,22,23 the medial smooth muscle of most large and medium-sized arteries appears to express only connexin43.10,24
In contractile smooth muscle of the normal vessel wall, gap junctions contribute to the modulation of vasomotor tone and maintenance of circulatory homeostasis.25 Evidence from in vitro studies suggests that gap junctions may also modulate cellular activities of significance in the pathogenesis of the vascular response to injury and disease. For example, coculture of SMCs with endothelial cells leads to both increased expression of connexin43 transcripts and growth factor production,26 and cultured synthetic phenotype SMCs show markedly enhanced connexin43 expression compared with their contractile counterparts.9,27 Moreover, in the human coronary artery, upregulation of connexin43 gap junctions between SMCs is a conspicuous early event in the development of atherosclerotic lesions.10
These findings raise key questions that have not previously been addressed. First, it is not yet known whether connexin43 gap junction upregulation in vivo is confined to arterial disease or constitutes an early component of the SMC synthetic transformation process in general. Second, if connexin43 gap junction upregulation were to prove to be of general significance in both arterial injury and disease, it would be important to establish its relationship to other key events, specifically migration of SMCs from media to intima and the timing of altered differentiation to the synthetic phenotype (eg, as reflected in altered expression of actin isoforms28,29). This information could shed light on such questions as whether gap-junctional communication is temporally linked to the initial synthetic transformation process and whether enhanced gap junction expression correlates with maintenance of the synthetic state.
To examine these issues, we investigated the spatial and temporal pattern of expression of connexin43 gap junctions between SMCs during vascular healing in the rat carotid artery after balloon catheter injury. Quantitative immunoconfocal microscopy was applied to localize and to quantify connexin43 gap junctions, and parallel studies were conducted to correlate changes in actin isoform expression to assess the state of cell differentiation. The rat balloon injury model provides a rapid method for the induction of intimal hyperplasia arising from phenotypic transformation of SMCs in vivo30 while providing sufficient temporal resolution to follow events that precede and culminate in neointimal formation. This model has previously been widely applied for examining the cellular events thought to contribute to postangioplasty restenosis8,31 and may also provide insights into fundamental aspects of SMC behavior relevant to the understanding of atherosclerosis.
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Methods |
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Samples and Tissue Processing
Adult male Sprague-Dawley rats (400 to 500 g) were anesthetized with sodium pentobarbital (20 mg/kg IP), and the left carotid artery was denuded of endothelium by the passage of a 2F balloon embolectomy catheter, leaving the uninjured right side as control. Animals were killed 1, 3, 9, and 14 days after balloon injury. Six animals were used at each time point except for the 9-day group, in which 5 animals were used (total, 23 rats). A few minutes before the animals were killed with an overdose of pentobarbital, they were injected intravenously (via the ear vein) with 100 IU/kg heparin to prevent intravascular blood clotting. Carotid arteries were dissected, flushed clear of blood with PBS, and immediately fixed for immunohistochemistry or electron microscopy. All procedures were conducted according to the Animals (Scientific Procedures) Act, 1986, under license from the Home Office.
For immunohistochemistry, arterial segments were fixed in 2% paraformaldehyde for 90 minutes at room temperature. After washing in buffer overnight, the samples were embedded in Tissue-Tek OCT and snap-frozen in isopentane held close to its freezing point (-160°C) using liquid nitrogen. Frozen sections (10 µm thick) of transverse rings of tissues were cut using a cryostat, then mounted on slides coated with Vectabond. Each slide had sections from both injured and uninjured arteries from the same animal.
For thin-section electron microscopy, selected samples were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.3) for 2 hours. The specimens were then rinsed in buffer, postfixed in cacodylate-buffered 2% OsO4, stained en bloc in uranyl acetate, dehydrated in ethanol, and embedded in epoxy resin by standard procedures. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 301 electron microscope.
Immunohistochemical Detection of Connexin43 Gap Junctions and
SMC 
/
-Actin
Anti-Connexin Antibodies
Two different antibodies that give identical labeling patterns
were used for the immunofluorescence detection of
connexin43, a mouse monoclonal antibody (Chemicon, Harrow, UK) and an
affinity-purified rabbit polyclonal antiserum (designated
C16).32,33 The former was raised against residues
252 to 270 and the latter against residues 314 to 322 of the rat
connexin43 sequence. In addition, we tested for the presence of
connexin40 and connexin37 using affinity-purified rabbit polyclonal
antisera raised against residues 254 to 268 of the rat connexin40
sequence and against residues 266 to 281 of the cytoplasmic C-terminal
tail of rat connexin37. The specificity of the antibodies was confirmed
by Western blotting and immunogold labeling (H.-I.Y. et al, unpublished
data, 1997).24
Anti-SMC /-Actin Antibody
Mouse anti–/-actin monoclonal antibody HHF35 (DAKO,
Wycombe, UK) was used to detect expression of /-actin by
SMCs.
Secondary Antibody/Detection Systems
The following secondary antibody/detection systems were used:
(1) biotinylated sheep anti-mouse or anti-rabbit immunoglobulin with
Texas Red–streptavidin (Amersham Life Science, Buckinghamshire, UK);
(2) donkey anti-mouse immunoglobulin conjugated to CY5 (Chemicon); and
(3) donkey anti-rabbit immunoglobulin conjugated to CY3 (Chemicon).
Visualization by Texas Red–streptavidin was used for standard labeling
and for semiquantitative analysis of connexin43 gap junctions
(see below). CY3 and CY5 were used for double labeling of connexin43
with SMC /-actin.
Immunolabeling of Connexin43 Gap Junctions
After air drying at room temperature, the sections were first
immersed in PBS for 5 minutes and treated for 15 minutes with 0.1%
Triton X-100 and 0.5% BSA, respectively. The sections were then
incubated with anti-connexin43 monoclonal antibody (1:1000) at room
temperature overnight. Slides were incubated in biotinylated sheep
anti-mouse antibody (1:250) at room temperature for 1 hour with
subsequent fluorescent visualization using Texas
Red–streptavidin (1:250). After final washing in PBS, the slides were
mounted using Citifluor mounting medium. Immunolabeling to test for
the presence of connexin40 or connexin37 followed a similar procedure
using the appropriate antibody (1:1000 dilution, 37°C for 30 minutes)
followed by biotinylated anti-rabbit/Texas Red–streptavidin. As
positive controls, rat left ventricular tissue was used,
with working myocardium serving as the positive control for
connexin43 and coronary artery endothelium for
connexins 40 and 37. Negative controls were done by (1) omitting the
primary antibody, (2) using an inappropriate secondary antibody, and
(3) in the case of polyclonal antibodies, peptide inhibition.
Double Labeling of Connexin43 With SMC /-Actin
After treatment with 0.1% Triton X-100 and 0.5% BSA, sections
were incubated sequentially with rabbit anti-connexin43 (C16) (1:50
dilution) at 37°C for 2 hours and HHF35 monoclonal antibody (1:50) at
room temperature for 1 hour. Mixed solutions of two secondary
antibodies, anti-rabbit CY3 (1:250) and anti-mouse CY5 (1:250), were
then applied at room temperature for 1 hour.
Sections of rat heart and aorta in the same run were used as positive
controls for immunolabeling of connexin43 and SMC /-actin,
respectively. Omission of primary antibodies was used as a negative
control.
Sections adjacent to those destined for semiquantification of gap junctions from both injured and uninjured vessels of the four groups were stained with the nucleus marker, ethidium bromide, at a concentration of 20 ng/cm3 in PBS at room temperature for 30 minutes to determine the cellularity of the medial layer and neointima (see below).
Confocal Laser Scanning Microscopy
Sections were examined by conventional epifluorescence
and confocal laser scanning microscopy (Leica TCS 4D, equipped with an
argon/krypton laser) fitted with the appropriate filter blocks for the
detection of fluorescein, Texas Red, Cy3, ethidium bromide,
and Cy5 fluorescence. For sections immunostained
with connexin43 monoclonal antibodies used for semiquantification of
gap junctions, the images were collected using the x63 objective lens
and the zoom 1.5 computer setting so that each pixel
represented 0.1 µm. Other settings were adjusted
according to instructions in the operating manual to optimize
recording of gap-junctional labeling. Once optimized, the
settings were kept constant for recording of data in the same
animal. For sections stained with ethidium bromide destined for
determination of cellularity, the x40 objective lens was used. Each
recorded image consisted of 1024 x 1024 pixels, and
projection views of three consecutive optical sections, separated
by 1 µm, were recorded for analysis. For double
labeling, the images were taken using simultaneous
dual-channel scanning. To detect the differential effects of balloon
injury across the vascular wall, elastic laminae were recorded
using the fluorescein isothiocyanate channel. All specimens
were examined within 24 hours of labeling.
Image Analysis and Semiquantification
Analysis and semiquantification of images from sections
labeled with the monoclonal anti-connexin43 antibody were undertaken
using PC image analysis software (Foster Findlay Associates,
Newcastle upon Tyne). Previous work has shown that the size of
immunofluorescent spots measured under defined conditions gives
a reliable indication of gap junction size as determined by electron
microscopic methods.34 For all groups, five
sections were taken from each sample. For the 1-, 3-, and 9-day groups,
four randomly selected fields containing the whole thickness of the
media were analyzed from the three technically best sections
from each animal. For the 14-day group, four randomly selected fields
from both the portion of media underlying well-developed
neointima and that with minimal or absent
neointima and four randomly selected fields containing the
whole thickness of neointima were analyzed for each
animal. Four randomly selected fields from the uninjured artery of each
animal were used as controls. Slides from all groups were processed
together with the observer blinded to the identity of each slide. A
binary overlay was created automatically. The arterial
medial layer was typically divided naturally into three zones, inner
(luminal facing), middle, and outer (adventitial facing), according to
the layers separated by elastic laminae, which were readily recognized
by the threshold setting because of their autofluorescence. Gap
junction analysis was conducted in each of these zones and in
the entire neointima. The threshold of the binary overlays
was adjusted, by comparing the original image on another monitor, to
make the optimal match of each spot. The value of the threshold setting
was kept constant in the same animal. After editing by hand to reject
autofluorescence, the number and dimensions of immunolabeled
gap junctions were calculated automatically. Occasionally, more than
four elastic laminae were present, giving more than three medial
layers in the media; in such cases, the portion beyond the second layer
from the intimal side was taken as the outer zone. Cellularity was
determined by counting the number of nuclei in each medial zone and in
the neointima, excluding ethidium bromide spots of less
than 7 µm diameter. Mean values (±SEM) of the longer diameter
of the immunolabeled gap junctions and of the area of immunolabeled gap
junction per unit volume of tissue sampled
(µm2/µm3) were
determined for each experimental group. To estimate the area of
immunolabeled gap junctions per cell, the latter was divided by the
cell number per unit volume of tissue (derived from the ethidium
bromide results). The data were compared statistically by Student's
t test.
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Results |
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The pattern of expression of connexin43 gap junctions in the arterial wall was markedly altered after balloon catheter injury. The sequence of changes observed is illustrated in Fig 1
, and the data are summarized in Fig 2. Connexin40 and connexin37 labeling,
although present in endothelium, were absent from
the medial smooth muscle of rat carotid artery.
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Connexin43 Gap Junction Distribution in the Normal (Noninjured)
Arterial Wall
Immunolabeled connexin43 gap junctions in control (noninjured)
arteries of all experimental groups showed a characteristic pattern in
the form of scattered, sharply defined spots throughout the media (Fig 1
, A). The distribution of the punctate label was
heterogeneous; the gap junctions were distributed both
singly and in clusters or chains, often with substantial intervening
areas devoid of label. Comparison of the data on immunolabeled gap
junction quantity from each of the individual zones of the controls
obtained at the different time intervals revealed no significant
differences (P>.1); the control values for each zone were
therefore pooled. When the gap junction values for the different zones
in these pooled controls were compared, a significantly higher
concentration of gap junction labeling was apparent in the adventitial
(outer) zone than in either the luminal-facing (inner) or middle zone
(P<.01) (Fig 2). No labeling was apparent in negative
controls.
Connexin43 Gap Junction Distribution After Balloon Catheter
Injury
The changes in gap junction distribution observed after injury
were apparent in both the media (Fig 1, B through F) and the
neointima (Fig 1, D through F) but were most prominent in
the latter.
Neointima
The neointima was first seen 9 days after injury as an
isolated small mass of cells bulging from the luminal side of the
internal elastic lamina (Fig 1, D). Abundant uniformly distributed gap
junctions were observed at this very early stage (Fig 1, D). By 14 days
postinjury, the neointima had developed as a crescent along
the internal elastic lamina. As the neointima grew, the
distribution of gap junctions became more scattered and irregular, but
a remarkably high abundance of gap junctions was maintained (Fig 1, E).
The frequency of immunolabeled gap junctions in the
neointima at 14 days postinjury was threefold greater than
that observed in the subjacent arterial media (Fig 2), a
highly significant difference (neointima versus media,
P<.001).
It should be noted that the rate and extent of development of the
neointima varied between animals; some developed much
thicker neointimas than others (Fig 1, E and F). At its
thickest, the neointima was wider than the media. The
frequency of immunolabeled connexin43 gap junctions was particularly
dense in the thicker portions of neointima. Clusters of
immunolabeled junctions were commonly observed in these zones. The
dimensions of the immunolabeled gap junctions were significantly larger
in the neointima than in the media (neointima,
0.49±0.01µm; media, 0.37±0.01µm; P<.05).
Consistent with the results from immunoconfocal
microscopy, electron microscopy revealed abundant gap junctions between
neointimal SMCs (Fig 3, A)
and fewer, smaller gap junctions between medial smooth muscle cells
(Fig 3, B). Typically, the neointimal gap junctions were
situated where a process from one cell interacted with a
neighboring cell (Fig 3). We never observed gap junctions between cell
types other than SMCs.
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Media
The changes in gap junction organization in the media, although
less obvious than those in the neointima, were nevertheless
consistent and clear-cut. One day after balloon dilatation,
labeling appeared patchy, as in the control group, but the quantity of
labeling increased in the luminal (inner) zone, and that in the
adventitial (outer) zone was reduced compared with controls, thereby
giving similar values for all three zones (Fig 1, B). By 3 days after
injury, the increase in gap junction labeling in the inner zone had
become significantly different from that of the controls
(P<.01). By day 9, however, as the neointima
was beginning to appear, the original pattern had become reestablished
(Fig 1, C) such that the outer zone had a significantly higher gap
junction concentration than the inner and middle zones
(P<.05), as in the controls. Fig 2 summarizes the
quantitative changes observed.
The distribution of immunolabeled connexin43 gap junctions in the inner
zone of the media lying adjacent to the neointima in the 9-
and 14-day specimens varied according to the thickness of the
neointima. Clusters of gap junctions were typical of medial
regions underlying thin intimal zones, whereas under thick intimal
regions, the gap junctions were more dispersed (Fig 1, E and F). At 14
days, the gap junction frequency in the middle zone was significantly
lower (P<.05) than that of the remaining zones (Fig 2). The
overall frequency of gap junctions in the regions of the media
underlying the neointima did not differ from that in medial
regions, where no neointima was present
(P>.05).
Estimation of Cellularity of the Arterial Media and
Neointima and Determination of Immunolabeled Connexin43 Gap
Junction Area Per Cell
To estimate the cellularity of the various zones, and thereby
derive comparative estimates of gap junction content per cell, we
applied ethidium bromide staining, which labels nuclei bright red and
readily countable. Nuclei in the medial layers typically appeared
spindle-shaped in both injured and uninjured samples, whereas those of
the neointima were predominantly discoid, probably because
of differences in cell orientation (Fig 4, A through D). Nuclear counts revealed
a trend toward declining cellularity with time after injury in the
media as a whole and in the individual medial zones except in the inner
zone at 1 and 3 days after injury. The neointima had a
comparable cellularity to that of uninjured media. Fig 5 summarizes the data on quantity of
immunolabeled gap junctions expressed as junction area per cell derived
from the nuclear counts. These data revealed that in the media as a
whole, there was an initial increase in gap junction content per cell
after injury, but this was followed by a mild decline toward control
values around day 9. A similar trend was apparent when each of the
three zones was analyzed individually. In line with the data in
Fig 2, the neointima had a significantly higher gap
junction content per cell than the media (P<.001).
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Distribution of SMC /-Actin in Relation to
Connexin43
In the control (noninjured) artery, SMC /-actin was evenly
distributed across the three medial zones (Fig 6, A), but at 1 day after injury, a less
prominent, attenuated labeling pattern across these zones had become
apparent (Fig 6, B). At 3 days after injury, a gradient was observed,
with the highest concentration toward the luminal zone (Fig 6, C and
D). By 9 days after injury, however, the luminal zone had lost much of
the labeling, while prominent but heterogeneous labeling
was reestablished in the middle and adventitial zones (Fig 6, E). This
pattern of medial labeling remained the same at 14 days after injury,
the neointima showing only weak labeling (Fig 6, F).
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Because the results had demonstrated distinct spatiotemporal changes in
the patterns of expression of connexin43 and SMC /-actin, double
labeling of these two components was carried out to permit
simultaneous visualization and thereby establish the
relationship between gap junction presence and altered /-actin
expression. Overall, there was a general tendency for gap junction
labeling to predominate in areas with weak or a lack of /-actin
signal, although gap junctions between cells showing stronger
/-actin signal was also apparent. This is illustrated in Fig 6, G
and H, from 14-day postinjury specimens; connexin43 immunolabeling is
seen in both the predominantly /-actin–negative regions of the
inner and middle medial zones and the /-actin–positive regions
of the outer (adventitial) zone. The lack of an absolute correlation
between connexin43 and /-actin expression is also apparent from
the observation that connexin43 labeling of control (noninjured) media
was associated with prominent /-actin–positive staining, whereas
the extensive connexin43 labeling of the neointima in
14-day animals was associated with very weak /-actin
staining.
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Discussion |
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A key finding of the present study is that connexin43 gap junctions are abundantly expressed between SMCs in the neointima formed after balloon injury in the rat carotid artery. Leading up to this event are more subtle alterations in medial SMC connexin43 expression, notably a transient increase in gap junction expression in the innermost (subluminal) zone, the major site from which the cells subsequently found in the neointima are recruited. These findings reinforce and significantly extend the results of our earlier studies on the relationship between arterial SMC phenotype, connexin43 expression, and the pathogenesis of proliferative intimal growth.
We previously reported that upregulation of connexin43 gap junction expression between SMCs correlates with phenotypic transition to the synthetic state in vitro9 and is prominent in the early thickened intimal lesions of human coronary atherosclerosis.10 The present findings, interpreted against this background, indicate that enhanced connexin43 expression by arterial SMCs in vivo is not confined to the slowly evolving intimal growth associated with atherosclerotic disease but is also a conspicuous event in the much more rapid intimal growth induced by balloon injury. Although the cellular events following mechanical injury in this rat model differ in detail from those observed in atherosclerosis, eg, the time course and relative contributions of intimal hyperplasia differ,6,35,36 and lipids play a key role only in the latter,37 the fundamental feature of SMC phenotypic change is common to both. By using an animal model of acute injury to induce SMC phenotypic change, it is possible to achieve a much more precise temporal and spatial resolution of changing connexin expression patterns than is possible in the study of human disease, where the nature of the samples precludes a true temporal pathogenetic sequence.
Our findings that an increase in SMC gap junctions is detectable on the luminal side of the media as early as 1 to 3 days after injury, that numerous gap junctions are apparent in the very earliest of neointima, and that the abundance of gap junctions is maintained in the well-developed neointima suggest that gap-junctional communication between SMCs may play a role both in the initiation of the proliferative response and subsequently in maintaining it. Although we do not know what triggers such a rapid upregulation of connexin43, synthetic-state cells are known to express growth factor receptors,38 and recent studies on cultured SMCs have shown that connexin expression is modulated by growth factors.11–13 Thrombin is a particularly potent simulator of connexin43 expression in cultured arterial SMCs13,39 and is a strong candidate for mediating the corresponding effect in vivo because sites denuded of endothelium are strongly prothrombogenic.
Our observation that connexin43 gap junctions in the media of the
uninjured artery are not uniformly distributed but show a higher
concentration toward the adventitial side is not surprising when set in
the context of the numerous physiological gradients
(eg, differences in pressure and access of blood supply), which operate
across the normal vascular wall. That the connexin43 immunolabeling
visualized by confocal microscopy here and in the injured
arterial wall is predominantly if not exclusively due to
SMC gap junctions is confirmed by our findings that (1)SMCs, and no
other cell types within the tissue, had morphologically defined gap
junctions on thin-section electron microscopy and (2) the abundance of
these ultrastructurally defined junctions correlated directly with the
extent of connexin43 labeling observed by confocal microscopy. Apart
from endothelial cells, which are removed on balloon
injury, the only other candidate connexin43-expressing cell types are
macrophages and other leukocytes. The former, although
reportedly having the capacity to express connexin43 mRNA and
protein,40,41 do not actually appear to form gap
junctions,42 and the latter do not normally form
gap junctions except possibly as a transient event with
endothelial cells.43 Moreover,
macrophages and other leukocytes account for only a small
percentage (
1%) of the total cell number in the
neointima of balloon-injured rat
carotid.44 Thus, the contribution of other cell
types to the immunoconfocal analysis of connexin43 label is
negligible and for practical purposes may be excluded.
In line with the identification of the connexin43 expression
specifically to SMCs, our double immunoconfocal labeling for connexin43
and smooth muscle /-actin showed many instances of
superimposition. This colocalization, however, was incomplete in the
injured samples, where considerable variation in the intensity of the
/-actin signal was apparent in the media, and the
neointima characteristically showed uniform weak labeling.
Decreased -actin and increased -actin expression are
well-documented features of the phenotypic transformation of SMCs from
the quiescent (contractile) state to the activated (synthetic)
state in animal models of atherosclerosis and vascular
injury29,45,46 and in human
atherosclerosis,47 the -actin
decline generally being much greater in magnitude than the -actin
increase. The relatively weak /-actin signal in the
neointimal SMCs and localized lack of staining in the media
are thus attributable to quantitative differences between the changing
levels of expression of the two isoforms in cells undergoing phenotypic
change. Abundant connexin43 labeling in the neointima,
where the /-actin signal is weak, is in keeping with our concept
that synthetic-state cells characteristically show enhanced connexin43
expression.10 There is, however, no absolute
correlation between the presence of gap junctions and the intensity of
/-actin signal.
A key question concerns the relationship between connexin43 upregulation and the migratory and proliferative properties associated with the synthetic phenotype in SMCs. The SMC response to injury in the rat carotid artery initially involves replication in the media, followed by migration to the neointima and subsequent replication in the neointima.48 Proliferation and migration are, however, separable events; although some subpopulations of cells undergo proliferation in the media before migration, others may migrate directly, without initial proliferation.49 To migrate, SMCs might be predicted to have to shed their links with their neighbors and hence lose their gap junctions. Cell division is also associated with loss of gap junctions in a number of cell types,50 and connexin43 gap junctions are downregulated in cultured SMCs expressing the proliferation markers PCNA and Ki67 (H-I Yeh, unpublished observations). In addition, reduced gap-junctional intercellular communication has been widely linked to rapid proliferation and uncontrolled growth; some cancer cells, eg, are reported to show gap junction loss, and transfection of connexins into transformed cells is reported to restore gap-junctional communication and to suppress cell and tumor growth in nude mice.17 From this background, lack of gap junctions, rather than the abundance observed, might have been expected to typify the synthetic-state SMC during rapid intimal growth.
Our present findings on the intact arterial wall suggest a rather different view, that upregulation of connexin43 gap junctions between SMCs, leading to increased capacity for direct intercellular signaling, is closely linked with SMC activation and early intimal growth. Gap junction upregulation may be a transient event in the cell cycle of the asynchronously dividing cells and/or a more permanent feature of specific subpopulations of synthetic cells that are neither actively migrating nor expressing proliferation markers. Precisely why the SMCs might need to communicate under these circumstances, the nature of the putative signaling molecules involved, and the significance of our findings in relation to hypotheses of growth control are not yet known. However, the concept of distinct SMC subpopulations is consistent with the observed heterogeneity in connexin43 gap junction distribution and with the identification of subgroups of cells differing in other characteristics, such as differentiation markers,51,52 platelet-derived growth factor B expression,53 and migratory and proliferative responses.49 Although connexin43 gap junction upregulation in SMCs is clearly a correlate of the synthetic phenotype and intimal growth, it should be emphasized that not all synthetic-state SMCs appear necessarily to have an abundance of connexin43 at all times. In advanced atherosclerotic lesions, the quantity of immunodetectable connexin43 between SMCs is markedly reduced,10 and SMCs isolated from such lesions show a lower capacity for intercellular communication.54
In conclusion, we show that connexin43 gap junction formation between SMCs participates in the early stages of the response of the vascular wall to balloon catheter injury. From our present and past findings, we hypothesize that early upregulation of connexin43 gap junctions may be a key event in the SMC phenotypic transition underlying the vascular response to injury and disease and may subsequently contribute to maintenance of the synthetic state. Notwithstanding the documented differences in the responses of the healthy versus diseased arterial wall to injury,6,35–37 our findings may prove relevant to our understanding of the cellular mechanisms underlying restenosis following balloon angioplasty and other interventions aimed at restoring patency in diseased arteries.
Note added in proof. A recent article by Polacek et al (J Vasc Res. 1997;34:19-30) reported equivalent levels of connexin43 expression in intimal and medial SMCs of the rabbit arterial wall after balloon injury, these levels being similar to those of medial SMCs of the noninjured artery. It should be noted that the postinjury time point in the study by Polacek et al is much later than the period examined in our study, and the SMCs they examined may well have reverted to the contractile phenotype.
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Acknowledgments |
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This work was supported in part by project grants from The Wellcome Trust (grant 046218/Z/95) and the British Heart Foundation (grant PG 93136). We thank Stephen Rothery for his expertise with confocal microscopy and figure preparation and Dr A. Esmail for assistance with the initial balloon catheter injury experiments.
Received April 4, 1997; accepted July 30, 1997.
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