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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3174-3184

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3174-3184.)
© 1997 American Heart Association, Inc.

Articles

Upregulation of Connexin43 Gap Junctions Between Smooth Muscle Cells After Balloon Catheter Injury in the Rat Carotid Artery

Hung-I Yeh; Florea Lupu; Emmanuel Dupont; ; Nicholas J. Severs

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.


*    Abstract
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 *Abstract
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 down arrowResults
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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


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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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.


*    Methods
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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 {alpha}/{gamma}-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 {alpha}/{gamma}-Actin Antibody
Mouse anti–{alpha}/{gamma}-actin monoclonal antibody HHF35 (DAKO, Wycombe, UK) was used to detect expression of {alpha}/{gamma}-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 {alpha}/{gamma}-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 {alpha}/{gamma}-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 {alpha}/{gamma}-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.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *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 1Down, and the data are summarized in Fig 2Down. Connexin40 and connexin37 labeling, although present in endothelium, were absent from the medial smooth muscle of rat carotid artery.



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  		Figure 1. Sequence of confocal images illustrating the changing pattern of connexin43 gap junction expression after balloon catheter injury of the rat carotid artery. In all these images, connexin43 gap junctions appear as red spots, the elastic laminae are seen as green striations, and the lumen (L) is at the top of the field. In the control (uninjured) artery (A), connexin43 gap junctions are heterogeneously scattered throughout the media, with an overall higher concentration toward the adventitial side. Initially, at 1 day postinjury (B), this interzone difference disappears but is reestablished by 9 days postinjury (C). At the 9-day stage, the first signs of neointimal formation (n) are apparent (D) as a small mass of cells on the luminal side of the internal elastic lamina, which contains abundant gap junctions. Examples of the variation in neointimal thickness at 14 days are shown in E and F; in all cases, an abundance of gap junctions was apparent. Bar=12 µm.



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  		Figure 2. Immunoconfocal analysis of gap junction area per unit volume of tissue after balloon injury in the rat carotid artery. The medial zones of the arterial wall defined by the elastic laminae were analyzed individually. Pooled data for the media as a whole and for the neointima are also shown. The control values shown were pooled from data obtained from time-matched controls after verifying that there were no significant differences between controls from the individual time points. A significantly greater density of gap junctions is present on the outermost (adventitial) side of the media in control and 9-day postinjury samples (P<.01 and 0.05, respectively). The difference between inner- and outer-zone values in the controls is significantly different from the 1-day injured samples (P<.05). By 3 days postinjury, a further increase in gap junction labeling occurs; the value observed in the luminal zone at this point is significantly different from that in the controls (P<.01). The neointima at 14 days postinjury has a remarkable abundance of gap junctions, at a density threefold greater and highly significantly different from that of the media (*P<.001).

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 1Up, 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 2Up). 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 1Up, B through F) and the neointima (Fig 1Up, 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 1Up, D). Abundant uniformly distributed gap junctions were observed at this very early stage (Fig 1Up, 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 1Up, 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 2Up), 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 1Up, 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 3Down, A) and fewer, smaller gap junctions between medial smooth muscle cells (Fig 3Down, B). Typically, the neointimal gap junctions were situated where a process from one cell interacted with a neighboring cell (Fig 3Down). We never observed gap junctions between cell types other than SMCs.



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  		Figure 3. Thin-section electron micrographs demonstrating typical gap junctions (gj) between SMCs of the neointima (A) and media (B). Gap junctions were observed only between SMCs, confirming that the connexin43 immunolabeling in Fig 1Up represents SMC gap junctions. Neointimal SMCs had abundant, large gap junctions, whereas medial SMCs had smaller, less frequent junctions, in accord with the confocal observations. The inset for each figure shows an enlarged view of the gap junctions in which the diagnostic pentalaminar structure is clearly seen. P indicates neointimal SMC process. Bar=0.5 µm; inset, x92,000.

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 1Up, 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 1Up, 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 2Up 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 1Up, 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 2Up). 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 4Down, 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 5Down 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 2Up, the neointima had a significantly higher gap junction content per cell than the media (P<.001).



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  		Figure 4. Ethidium bromide staining of nuclei visualized by confocal microscopy, used to estimate cellularity and to derive estimates of immunostained gap junction area per cell (Fig 6Up). Note that the cellularity of the inner (luminal layer) is increased at 1 and 3 days postinjury. N indicates neointima. Bar=20 µm.



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  		Figure 5. Immunoconfocal analysis of gap junction area per cell. The data follow a similar pattern to that observed for gap junction area per unit volume of tissue (Fig 2Up).

Distribution of SMC {alpha}/{gamma}-Actin in Relation to Connexin43
In the control (noninjured) artery, SMC {alpha}/{gamma}-actin was evenly distributed across the three medial zones (Fig 6Down, A), but at 1 day after injury, a less prominent, attenuated labeling pattern across these zones had become apparent (Fig 6Down, B). At 3 days after injury, a gradient was observed, with the highest concentration toward the luminal zone (Fig 6Down, 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 6Down, E). This pattern of medial labeling remained the same at 14 days after injury, the neointima showing only weak labeling (Fig 6Down, F).



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  		Figure 6. Immunoconfocal visualization of {alpha}/{gamma}-actin expression in medial and neointimal SMCs in relation to connexin43 gap junctions. A through F show changes in {alpha}/{gamma}-actin expression, as seen by single labeling. Strong signal is apparent in control samples (A) because of the abundance of {alpha}-actin in the contractile phenotype. Altered staining patterns are apparent as the cells undergo phenotypic alteration to the synthetic state (B through F). SMCs in the neointima (n), although stained with the antibody, typically show weaker signal than contractile-state cells (F). Double immunoconfocal detection for {alpha}/{gamma}-actin (red fluorescence) and gap junctions (green fluorescent dots, arrows) is shown in G and H. Here, the elastic laminae are shown in blue to distinguish them from the gap junctions. Gap junctions are present in both areas strongly stained for {alpha}/{gamma}-actin (+) and those weakly stained for {alpha}/{gamma}-actin (*). Bar=20 µm.

Because the results had demonstrated distinct spatiotemporal changes in the patterns of expression of connexin43 and SMC {alpha}/{gamma}-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 {alpha}/{gamma}-actin expression. Overall, there was a general tendency for gap junction labeling to predominate in areas with weak or a lack of {alpha}/{gamma}-actin signal, although gap junctions between cells showing stronger {alpha}/{gamma}-actin signal was also apparent. This is illustrated in Fig 6Up, G and H, from 14-day postinjury specimens; connexin43 immunolabeling is seen in both the predominantly {alpha}/{gamma}-actin–negative regions of the inner and middle medial zones and the {alpha}/{gamma}-actin–positive regions of the outer (adventitial) zone. The lack of an absolute correlation between connexin43 and {alpha}/{gamma}-actin expression is also apparent from the observation that connexin43 labeling of control (noninjured) media was associated with prominent {alpha}/{gamma}-actin–positive staining, whereas the extensive connexin43 labeling of the neointima in 14-day animals was associated with very weak {alpha}/{gamma}-actin staining.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {alpha}/{gamma}-actin showed many instances of superimposition. This colocalization, however, was incomplete in the injured samples, where considerable variation in the intensity of the {alpha}/{gamma}-actin signal was apparent in the media, and the neointima characteristically showed uniform weak labeling. Decreased {alpha}-actin and increased {gamma}-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 {alpha}-actin decline generally being much greater in magnitude than the {gamma}-actin increase. The relatively weak {alpha}/{gamma}-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 {alpha}/{gamma}-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 {alpha}/{gamma}-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.


*    Acknowledgments
 
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.


*    References
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  1. Stary HC, Blankenhorn DH, Chandler AB, et al. Special report: a definition of the intima of human arteries and of its atherosclerosis-prone regions: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1992;85:391–405.[Free Full Text]
  2. Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty: potential biologic determinants and role of intimal hyperplasia. Circulation. 1989;79:1374–1387.[Abstract/Free Full Text]
  3. Bai H, Masuda J, Sawa Y, et al. Neointima formation after vascular stent implantation—spatial and chronological distribution of smooth muscle cell proliferation and phenotypic modulation. Arterioscler Thromb. 1994;14:1846–1853.[Abstract/Free Full Text]
  4. Macleod DC, Strauss BH, de Jong M, et al. Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions. J Am Coll Cardiol. 1994;23:59–65.[Abstract]
  5. Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995;75:487–517.[Abstract/Free Full Text]
  6. Schwartz SM, Reidy MA, O'Brien ERM. Assessment of factors important in atherosclerotic occlusion and restenosis. Thromb Haemost. 1995;74:541–551.[Medline] [Order article via Infotrieve]
  7. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol. 1995;57:791–804.[Medline] [Order article via Infotrieve]
  8. Schwartz SM, DeBlois D, O'Brien ERM. The intima—soil for atherosclerosis and restenosis. Circ Res. 1995;77:445–465.[Free Full Text]
  9. Rennick RE, Connat J-L, Burnstock G, Rothery S, Severs NJ, Green CR. Expression of connexin43 gap junctions between cultured vascular smooth muscle cells is dependent upon phenotype. Cell Tissue Res. 1993;271:323–332.[Medline] [Order article via Infotrieve]
  10. Blackburn JP, Peters NS, Yeh H-I, Rothery S, Green CR, Severs NJ. Upregulation of connexin43 gap junctions during early stages of human coronary atherosclerosis. Arterioscler Thromb Vasc Biol. 1995;15:1219–1228.[Abstract/Free Full Text]
  11. Mensink A, De Haan LHJ, Lakemond CMM, Koelman CA, Koeman JH. Inhibition of gap junctional intercellular communication between primary human smooth muscle cells by tumor necrosis factor {alpha}. Carcinogenesis. 1995;16:2063–2067.[Abstract/Free Full Text]
  12. Mensink A, Brouwer A, Van den Burg EH, et al. Modulation of intercellular communication between smooth muscle cells by growth factors and cytokines. Eur J Pharmacol. 1996;310:73–81.[Medline] [Order article via Infotrieve]
  13. Yeh H-I, Kanthou C, Dupont E, Lupu F, Severs NJ. Differential effects of growth factors on gap junction expression in cultured human aortic smooth muscle cells. Eur Heart J. 1996;17:397. Abstract.
  14. Kumar NM, Gilula NB. The gap junction communication channel. Cell. 1996;84:381–388.[Medline] [Order article via Infotrieve]
  15. Gros DB, Jongsma HJ. Connexins in mammalian heart function. Bioessays. 1996;18:719–730.[Medline] [Order article via Infotrieve]
  16. Severs NJ, Dupont E, Kaprielian RR, Yeh H-I, Rothery S. Gap junctions and connexins in the cardiovascular system. In: Yacoub MH, Carpentier A, Pepper J, Fabiani J-N, eds:. Annual of Cardiac Surgery 1996, 9th ed. London, UK: Current Science; 1996:31–44.
  17. Yamasaki H, Naus CCG. Role of connexin genes in growth control. Carcinogenesis. 1996;17:1199–1213.[Free Full Text]
  18. White TW, Bruzzone R. Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences. J Bioenerg Biomembr. 1996;28:339–350.[Medline] [Order article via Infotrieve]
  19. Elfgang C, Eckert R, Lichtenberg-Fraté H, et al. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol. 1995;129:805–817.[Abstract/Free Full Text]
  20. Veenstra RD, Wang HZ, Beblo DA, et al. Selectivity of connexin-specific gap junctions does not correlate with channel conductance. Circ Res. 1995;77:1156–1165.[Abstract/Free Full Text]
  21. White TW, Paul DL, Goodenough DA, Bruzzone R. Functional analysis of selective interactions among rodent connexins. Mol Biol Cell. 1995;6:459–470.[Abstract]
  22. Little TL, Beyer EC, Duling BR. Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am J Physiol Heart Circ Physiol. 1995;268:H729–H739.[Abstract/Free Full Text]
  23. Moore LK, Burt JM. Selective block of gap junction channel expression with connexin-specific antisense oligodeoxynucleotides. Am J Physiol Cell Physiol. 1994;267:C1371–C1380.[Abstract/Free Full Text]
  24. Yeh H-I, Dupont E, Coppen S, Rothery S, Severs NJ. Gap junction localization and connexin expression in cytochemically identified endothelial cells from arterial tissue. J Histochem Cytochem. 1997;45:539–550.[Abstract/Free Full Text]
  25. Christ GJ, Spray DC, El-Sabban M, Moore LK, Brink PR. Gap junctions in vascular tissues—Evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ Res. 1996;79:631–646.[Abstract/Free Full Text]
  26. Navab M, Ross LA, Hama S, et al. Interactions of human aortic wall cells in co-culture. Atheroscler Rev. 1991;23:153–160.
  27. Lash JA, Critser ES, Pressler ML. Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos. J Biol Chem. 1990;265:13113–13117.[Abstract/Free Full Text]
  28. Orlandi A, Ehrlich HP, Ropraz P, Spagnoli LG, Gabbiani G. Rat aortic smooth muscle cells isolated from different layers and at different times after endothelial denudation show distinct biological features in vitro. Arterioscler Thromb. 1994;14:982–989.[Abstract/Free Full Text]
  29. Kocher O, Gabbiani F, Gabbiani G, et al. Phenotypic features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening: biochemical and morphologic studies. Lab Invest. 1991;65:459–470.[Medline] [Order article via Infotrieve]
  30. Thyberg J, Blomgren K, Hedin U, Dryjski M. Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study. Cell Tissue Res. 1995;281:421–433.[Medline] [Order article via Infotrieve]
  31. Clowes AW, Clowes MM, Fingerle J, Reidy MA. Kinetics of cellular proliferation after arterial injury: V. Role of acute distension in the induction of smooth muscle proliferation. Lab Invest. 1989;60:360–364.[Medline] [Order article via Infotrieve]
  32. El Aoumari A, Fromaget C, Dupont E, et al. Conservation of a cytoplasmic carboxy-terminal domain of connexin43, a gap-junctional protein, in mammalian heart and brain. J Membr Biol. 1990;115:229–240.[Medline] [Order article via Infotrieve]
  33. Jou YS, Dupont E, Lu SC, et al. Restoration of gap junctional intercellular communication in a communication deficient rat liver cell mutant by transfection with connexin 43 cDNA. Mol Carcinog. 1993;8:234–244.[Medline] [Order article via Infotrieve]
  34. Green CR, Peters NS, Gourdie RG, Rothery S, Severs NJ. Validation of immunohistochemical quantification in confocal scanning laser microscopy: a comparative assessment of gap junction size with confocal and ultrastructural techniques. J Histochem Cytochem. 1993;41:1339–1349.[Abstract]
  35. Strauss BH, Umans VA, Van Suylen RJ, et al. Directional atherectomy for treatment of restenosis within coronary stents: clinical, angiographic, and histologic results.J Am Coll Cardiol. 1992;20:1465–1473.
  36. Geary RL, Williams JK, Golden D, Brown DG, Benjamin ME, Adams MR. Time course of cellular proliferation, intimal hyperplasia, and remodeling following angioplasty in monkeys with established atherosclerosis—a nonhuman primate model of restenosis. Arterioscler Thromb Vasc Biol. 1996;16:34–43.[Abstract/Free Full Text]
  37. Recchia D, Abendschein DR, Saffitz JE, Wickline SA. The biologic behavior of balloon hyperinflation-induced arterial lesions in hypercholesterolemic pigs depends on the presence of foam cells. Arterioscler Thromb Vasc Biol. 1995;15:924–929.[Abstract/Free Full Text]
  38. Saltis J, Thomas AC, Agrotis A, Campbell JH, Campbell GR, Bobik A. Expression of growth factor receptors in arterial smooth muscle cells—dependency on cell phenotype and serum factors. Atherosclerosis. 1995;118:77–87.[Medline] [Order article via Infotrieve]
  39. Dupont E, Yeh H-I, Kanthou C, Kakkar VV, Lupu F, Severs NJ. Rapid modulation of gap junction expression by thrombin in cultured human smooth muscle cells. Eur Heart J. 1996;17:197. Abstract.
  40. Beyer EC, Steinberg TH. Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J Biol Chem. 1991;266:7971–7974.[Abstract/Free Full Text]
  41. Polacek D, Lal R, Volin MV, Davies PF. Gap junctional communication between vascular cells: induction of connexin43 messenger RNA in macrophage foam cells of atherosclerotic lesions. Am J Pathol. 1993;142:593–606.[Abstract]
  42. Alves LA, Coutinho-Silva R, Persechini PM, Spray DC, Savino W, De Carvalho ACC. Are there functional gap junctions or junctional hemichannels in macrophages? Blood. 1996;88:328–334.[Abstract/Free Full Text]
  43. Jara PI, Boric MP, Sáez JC. Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia–reperfusion. Proc Natl Acad Sci U S A. 1995;92:7011–7015.[Abstract/Free Full Text]
  44. Ferns GAA, Reidy MA, Ross R. Balloon catheter de-endothelialization of the nude rat carotid. Am J Pathol. 1991;138:1045–1057.[Abstract]
  45. Thyberg J, Hedin U, Sjolund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis. 1990;10:966–990.[Free Full Text]
  46. Desmoulière A, Gabbiani G. The cytoskeleton of arterial smooth muscle cells during human and experimental atheromatosis. Kidney Int. 1992;41(suppl 37):87–89.
  47. Glukhova MA, Kabakov AE, Frid MG, et al. Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci U S A. 1988;85:9542–9546.[Abstract/Free Full Text]
  48. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
  49. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res. 1985;56:139–145.[Abstract/Free Full Text]
  50. Dermietzel R, Yancey SB, Traub O, Willecke K, Revel J-P. Major loss of the 28-kd protein of gap junction in proliferating hepatocytes. J Cell Biol. 1987;105:1925–1934.[Abstract/Free Full Text]
  51. Gabbiani G, Rungger-Brändle E, De Chastonay C, Franke WW. Vimentin-containing smooth muscle cells in aortic intimal thickening after endothelial injury. Lab Invest. 1982;47:265–269.[Medline] [Order article via Infotrieve]
  52. Zanellato AMC, Borrione AC, Tonello M, Scannapieco G, Pauletto P, Sartore S. Myosin isoform expression in smooth muscle cell heterogeneity in normal and atherosclerotic rabbit aorta. Arteriosclerosis. 1990;10:996–1009.[Abstract/Free Full Text]
  53. Lindner V, Giachelli CM, Schwartz SM, Reidy MA. A subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-B chain mRNA. Circ Res. 1995;76:951–957.[Abstract/Free Full Text]
  54. Andreeva ER, Serebryakov VN, Orekhov AN. Gap junctional communication in primary culture of cells derived from human aortic intima. Tissue Cell. 1995;27:591–597.[Medline] [Order article via Infotrieve]



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