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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1219-1228.)
© 1995 American Heart Association, Inc.

Articles

Upregulation of Connexin43 Gap Junctions During Early Stages of Human Coronary Atherosclerosis

J. P. Blackburn; N. S. Peters; H.-I. Yeh; S. Rothery; C. R. Green; N. J. Severs

From the Department of Cardiac Medicine, National Heart and Lung Institute, London, England (J.P.B., H.-I.Y., S.R., N.J.S.); Department of Cardiology, St Mary's Hospital Medical School, London (N.S.P.); and Department of Anatomy, School of Medicine, University of Auckland, New Zealand (C.R.G.).

Correspondence to Professor N.J. Severs, Department of Cardiac Medicine, National Heart and Lung Institute, Royal Brompton Hospital, Sydney St, London SW3 6NP, England.

	Abstract

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Abstract Interactions between cells form the framework for understanding the pathogenesis of atherosclerosis, but little information is available on the role of direct intercellular communication via gap junctions in this process. To investigate gap junction expression in the pathogenesis of human atherosclerosis, lesions representing different stages of the disease were obtained from coronary arteries of hearts removed from patients undergoing cardiac transplantation. Twelve hearts, each providing 1 to 3 segments of artery, were used in the study. Sections were examined by confocal laser scanning microscopy after immunofluorescent labeling with a specific antibody against connexin43, the major gap-junctional protein of smooth muscle cells, to permit high-definition visualization of immunolabeled gap junctions through the depth of the specimen. Double labeling using anti-connexin43 and cell type–specific antibodies demonstrated colocalization of gap junctions with smooth muscle cells but not with macrophages, a relationship confirmed by electron microscopy. Regions of intimal thickening and early atheromatous lesions showed markedly increased expression of connexin43 gap junctions between intimal smooth muscle cells compared with the undiseased vessel. This increase in gap junctions was most marked in regions of intimal thickening, semiquantitative analysis of the confocal digital images revealing a >10-fold increase compared with the undiseased vessel. The quantity of labeled gap junctions in early atheromatous lesions, although higher than that of the undiseased vessel, was lower than that of intimal thickenings, and this trend toward reduced levels of gap junction immunolabeling with lesion progression continued, the value observed in the most advanced atheromatous lesions being lower than that of the undiseased vessel. As the quantity of gap junctions declined, their distribution became more patchy and the sizes of individual junctions larger. The results suggest that enhanced expression of gap junctions between smooth muscle cells may play a role in maintaining the synthetic phenotype during early growth of the atherosclerotic plaque.

Key Words: gap junctions • connexin43 • smooth muscle cell • atheroma • coronary artery

	Introduction

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Atherosclerosis is a multifactorial disease process initiated by a variety of pathogenetic mechanisms that converge on a common target: altered behavior in the arterial smooth muscle cell.^{1 2 3 4} Smooth muscle cells in the media of the healthy artery are differentiated to fulfill a contractile function, but under the influence of chemoattractants and mitogens, they can be induced to migrate into the intima, proliferate, and become highly active synthetically.⁵ The massive quantities of extracellular material produced by smooth muscle cells that have undergone such phenotypic transformation to the synthetic state are responsible for the bulk growth of the atherosclerotic lesion.

Initiation and progression of the atherosclerotic plaque involves complex patterns of interaction between the cells of the arterial wall, in which growth factors and cytokines are known to play a critical role.^{1 2 3 4 6 7} Apart from such well-established extracellular signaling mechanisms, another form of interaction between cells involves direct intercellular communication via specialized interacting domains of the plasma membrane called gap junctions. Gap junctions consist of clusters of channels that bridge the apposing plasma membranes of neighboring cells, directly linking their cytoplasmic compartments.^{8 9 10 11 12 13 14} Each gap-junctional channel consists of a pair of connexons ("hemichannels"), one contributed by each of the junctional membranes, the connexon itself being constructed from six connexin monomers. The connexins are a multigene family of conserved proteins, different members of which are expressed in different cell types, tissues, and species.^{11 15 16}

The channels formed by gap junctions permit direct cell-to-cell passage of ions and small molecules (<1 kD), thereby mediating intercellular metabolic and electrical coupling.^{8 9 10 12 13 14 17} Gap junctions are ubiquitous in multicellular systems, and gap-junctional intercellular communication has been implicated in a variety of biological functions, in particular in the regulation of growth, proliferation, differentiation, and development.^{18 19 20 21 22} Recent in vitro studies suggest that direct intercellular interaction via gap junctions may modulate cellular activities of significance in the pathogenesis of atherosclerosis.^{23 24} For example, coculture of smooth muscle cells with endothelial cells leads to both increased growth factor production and enhanced expression of messenger RNA encoding the gap-junctional protein connexin43.^{23 25 26} The level of connexin43 mRNA in cultured synthetic phenotype smooth muscle cells is approximately six times greater than that expressed in smooth muscle cells of the intact aorta,²⁷ and expression of the protein detectable by immunohistochemistry is high in the synthetic state but becomes markedly reduced in the contractile state.²⁸

Although these in vitro studies raise the possibility that gap-junctional communication may participate in the cellular interactions involved in atherogenesis, their precise significance has been difficult to assess because there has been no information to date on gap junction expression in the intact human arterial wall. The present study therefore set out to establish the pattern of smooth muscle connexin43 gap junction expression during human atherogenesis. To this end, we applied a specific antibody that we produced against connexin43,²⁹ combined with the technique of confocal laser scanning microscopy, to provide high-definition imaging of gap junctions in human arterial lesions.³⁰ Using samples of coronary arteries obtained from explanted hearts of transplant patients, we were able to collect lesions representing different stages of the disease and demonstrate that a distinctive spatial and temporal pattern of connexin43 expression between smooth muscle cells accompanies development of the atherosclerotic plaque.

	Methods

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Patients and Tissue Samples
Human coronary artery specimens were obtained from the explanted hearts of patients with end-stage ischemic heart disease or dilated cardiomyopathy who were undergoing cardiac transplantation. Upon ischemic arrest and removal of the heart, segments of coronary artery were immediately dissected out in the operating theater and placed in Zamboni's fixative,³¹ consisting of 2% paraformaldehyde/0.2% picric acid in 0.1 mol/L PBS (pH 7.4). Up to three segments per heart were obtained from different coronary arteries. From each segment, one 3-mm-thick transverse arterial ring was selected for study. Selected specimens were divided to provide samples for both confocal immunohistochemistry plus conventional histology and thin-section electron microscopy, with the orientation of the two arterial segments noted to enable subsequent matching of confocal image data with the ultrastructure. Samples destined for thin-section electron microscopy were transferred as quickly as possible from the Zamboni's fixative and placed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.3).

Tissue Processing
Samples for confocal microscopy were fixed for 2 to 4 hours in Zamboni's solution, washed in buffer overnight, dehydrated via a graded ethanol series, placed in chloroform overnight, and embedded in wax by standard histological procedures. For electron microscopy, fixation in glutaraldehyde was continued for 2 hours. The specimens were then rinsed in buffer, postfixed in 2% OsO₄, en bloc stained in uranyl acetate, dehydrated in ethanol, and embedded in epoxy resin by standard procedures.³² Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 301 electron microscope.

Immunohistochemical Detection of Gap Junctions and Identification of Cell Types
Antibodies
The primary polyclonal antiserum used for gap junction detection was raised against a synthetic peptide (designated "HJ") corresponding to residues 131 through 142 of the cytoplasmic loop of the transmembrane gap-junctional protein molecule connexin43.³³ The peptide was cross-linked to the carrier protein keyhole limpet hemocyanin with glutaraldehyde, and polyclonal antisera were raised in Sandy half-lop rabbits.^{29 34} Screening of the sera for reactivity was done by dot blot assay, and specificity to the corresponding peptide antigen was confirmed in dot blots. Specificity of the antiserum for gap-junctional protein was demonstrated by Western blotting, and specificity for ultrastructurally visualized gap junctions was shown by preembedment immunogold labeling of isolated cardiac gap junctions and by postembedment immunogold labeling of Lowicryl-embedded myocardial tissue.^{29 30 35} A related antiserum (designated "HH") that was raised against residues 101 through 112 of the connexin43 molecule²⁹ but that does not label gap junctions in sections of wax-embedded material³⁶ was used as a control.

Two monoclonal antibodies (purchased from Dako) were used in double-labeling experiments as specific cell-type markers: HAM56 for the detection of macrophages and HHF35 (an anti–/-actin antibody) for the detection of smooth muscle cells.^{25 26}

The secondary antibody/detection systems used in the study were as follows: (1) swine anti-rabbit TRITC, (2) goat anti-mouse FITC (both from Dako), and (3) biotinylated sheep anti-rabbit immunoglobulin and Texas Red–streptavidin (Amersham Life Sciences).

Immunolabeling of Gap Junctions
Transverse sections (10 µm thick) of wax-embedded arterial tissue were dewaxed in xylene and rehydrated in a graded ethanol series. For standard labeling of gap junctions, the sections were first incubated in a trypsin solution (containing 0.1% trypsin [Sigma T-8128], 0.1% CaCl₂, 20 mmol/L Trizma base, pH 7.4) for 10 minutes at room temperature to reexpose antigenic sites altered by fixation and processing.³⁷ After washing in buffer and treatment for 40 minutes in PBS containing 0.1 mol/L L-lysine (blocking agent) and 0.1% Triton X-100 (which acts as a wetting agent and further aids antigen exposure), the sections were incubated with the primary antiserum (dilution 1:10 in PBS) for 1 hour at 37°C. This was followed with a PBS wash and treatment with swine anti-rabbit TRITC (1:20 dilution) for 1 hour in the dark at room temperature. After final washing in PBS, the slides were mounted with Citifluor mounting medium (Citifluor Ltd). Immunolabeled sections were examined by conventional epifluorescence light microscopy and by confocal laser scanning microscopy. As positive controls, longitudinal sections of 70-day-old rat cardiac ventricle were included in each immunolabeling run. For negative controls, the HH antiserum described above was used in place of HJ.

Double Labeling of Gap Junctions With Specific Cell-Type Markers
Double-labeling experiments to enable simultaneous visualization of gap junctions with (1) smooth muscle cells and (2) macrophages were carried out by a procedure modified from that above. Sections were labeled sequentially with HJ antibody (1:100) followed by either HHF35 (for smooth muscle cell labeling) or HAM56 (for macrophage labeling) at a concentration of 1:50 (1 hour, room temperature). A buffer wash was given between and after the two antibody treatments. The sections were then exposed to two secondary antibodies: biotinylated sheep anti-rabbit immunoglobulin (1:250, 1 hour, room temperature; for the detection of HJ-labeled gap junctions) and goat anti-mouse FITC (1:25, 40 minutes, room temperature; for the detection of cell markers). Texas Red–streptavidin (1:250) was used for fluorescent visualization of the antibody-labeled gap junctions.

Confocal Laser Scanning Microscopy and Semiquantitative Analysis
Confocal microscopy was done with either a Bio-Rad Lasersharp MRC-500 or a Leica TCS 4D fitted with the appropriate filter blocks (for the detection of rhodamine, fluorescein, and Texas Red fluorescence). Double-labeled preparations were imaged with the latter instrument, equipped with an argon/krypton laser. The images were taken by simultaneous dual-channel scanning. The major part of the study (single labeling of gap junctions) was done with the Bio-Rad, with the confocal aperture set to 45% to 50% of its full adjustable range during image collection. Gain and contrast levels were set according to procedures standardized to ensure that the image collected demonstrated a full range of gray levels from black (0-pixel intensity level) to peak white (255-pixel intensity level). All sections were initially surveyed at low magnification with various objectives so that the overall distribution of fluorescent label could be assessed. Selected areas were subsequently examined at higher magnification using a x60 objective (Nikon, NA 1.4) and the zoom 1 setting on the computer (field size, 180x120 µm). Images were routinely Kalman-averaged to reduce background noise levels. All specimens were examined within 24 hours of immunolabeling.

The sizes of immunolabeled gap junctions were measured directly from the image data. Previous work has shown that immunofluorescent spot size measured under the specific conditions used correlates well with gap junction size determined by ultrastructural methods.³⁸ For semiquantitative comparative analysis of the quantity of gap junctions, the specimens were classified into four groups (representing different stages of disease; see below). Within each group, multiple specimens, each providing 10 randomly selected optical sections, were analyzed. Gap junction area per unit area of these samples (µm²x10^-5 per µm² of section) was determined by use of PC IMAGE image analysis software (Foster Findlay Associates), after fluorescence judged to be non–gap junctional in origin (eg, autofluorescence from elastic laminae) was edited out. The mean values (±SEM) were determined for each disease group and compared statistically by t test. It should be emphasized that the data obtained do not provide absolute values of gap-junctional content and are subject to a number of technical limitations. They may, however, be regarded as useful estimates of the relative quantities of gap junctions between groups for comparative purposes.

Correlative Histology and Classification of Lesions
Sections adjacent to those used for immunolabeling, stained with hematoxylin and eosin, were examined by standard bright-field optics for correlative histopathological assessment of each vessel and classification of lesion types. Assessment and identification were performed according to the criteria established by recognized authorities.^{39 40 41 42} To simplify presentation of the results, lesions were divided into four categories: (1) undiseased, (2) intimal thickening, (3) early atheroma, and (4) advanced atheroma. Group 2 includes the initial, fatty-streak, and intermediate lesions of atherosclerosis, as defined in a recent American Heart Association Scientific Statement.^{42 43} The third and fourth categories included all overt atherosclerotic lesions, including those commonly described as atheroma, fibrolipid plaque, and fibroatheroma, the distinction between these groups being made on the basis of the degree of occlusion of the vessel (group 3, <30% occluded; group 4, 30% occluded). Complicated lesions all fell within the fourth category. Specific types of lesion within the categories are described in more detail when relevant to interpretation and explanation of the findings. In groups 1 through 3, two zones are recognized within the intima: the proteoglycan layer on the endothelial side and the deeper musculoelastic layer.⁴⁴

	Results

Top Abstract Introduction Methods Results Discussion References

 
Human artery specimens were obtained from a total of 12 patients. Eleven were from coronary arteries of men (mean age±SD, 52±1.9 years); in addition, a diseased segment of internal mammary artery was obtained from the single female patient (age, 69 years). From these specimens, lesions representing various stages of the atherosclerotic disease process were obtained. Correlation of confocal immunolabeling results with standard histological appearance made it possible to follow the changing patterns of gap junction expression during the evolution of the atherosclerotic plaque. A schematic summary of these changes in distribution and abundance of gap junctions is presented in Fig 1.

View larger version (38K): [in this window] [in a new window]   Figure 1. Schematic summarizing the changes observed in connexin43-gap junction quantity, distribution, and size in relation to the principal events in the pathogenesis of atherosclerosis.

Figs 2 through 4 illustrate representative details of confocal immunolabeling results from three advanced lesions (category 4), set within the context of their overall histological appearance. Lesions in this category share a number of common features (marked luminal narrowing, extensive fibrous material, and variable amounts of lipid deposition) but show considerable variation in detailed histological morphology. They may, for example, comprise eccentric, solid fibrous plaques with finely dispersed lipid and crystalline cholesterol (Fig 2A), show extensive thrombus and almost totally occlude the vessel (Fig 3A), or appear as less obstructive fibrous plaques (Fig 4A). Confocal microscopy revealed positive immunolabeling of gap junctions as distinct fluorescent spots in all such lesions. Fig 2B illustrates a typical example, taken from the superficial intimal region indicated in the corresponding histological section in Fig 2A. Most of the samples revealed autofluorescent material that was distinguished from specific immunofluorescent label by alternating rhodamine and fluorescein filter blocks on the Nikon epifluorescence microscope. Specific label was seen only at the rhodamine wavelength, whereas the autofluorescent material had broader excitation and/or emission wavelengths and could be detected by use of either filter block.

View larger version (165K): [in this window] [in a new window]   Figure 2. Figs 2 through 4. Advanced atheroma. Examples of results obtained from three lesions (Figs 2, 3, and 4). The standard histology of each lesion (hematoxylin and eosin–stained wax section) is shown to the left (Figs 2A, 3A, and 4A), with examples of corresponding confocal scanning immunofluorescence images to the right in each case. The positions from which the confocal images are taken are marked on the histological sections with open arrows labeled with the letter of the corresponding figure panel. Immunolabeled gap junctions appear as sharp spots of fluorescence (examples indicated by arrows on 2B, 3B, 4B, and 4C). Note patchy distribution of gap junctions throughout the intima (Int) in Figs 2B, 3B, and 4B. Wavy zones of less intense autofluorescence (Figs 3B and 4C) represent elastic laminae (el). The media (M) shows relatively little gap junction immunolabeling (Figs 3B and 4C). L indicates lumen of vessel. Bar=1 mm for 2A, 3A, and 4A; Bar=20 µm for 2B, 3B, 4B, and 4C.

All advanced lesions showed a patchy distribution of moderately large (1 to 2 µm) immunolabeled gap junctions. The mean quantity of gap-junctional fluorescent spots measured in the advanced lesions was 6.2x10^-5 µm²/µm² section area (±1.6 SEM; n=60). Some variation in the extent of the gap junction labeling was apparent, according to specific features of the lesion. Specifically, there was an inverse correlation between the degree of fibrous deposition and the extent of gap junction immunolabeling, which may be seen by comparison of Figs 2 through 4. In the most advanced stages of fibroatheroma (such as that illustrated in Figs 2 and 3), immunolabeling was observed in the fibrous cap as small, sparsely scattered fluorescent spots (Fig 2B). In the deeper zones of the intima bordering the media (Fig 3B), the immunolabeled junctions were clustered to form discrete but relatively infrequently observed patches of label. In less occlusive lesions (Fig 4), immunolabeled gap junctions were more abundant. In such lesions, a distinct difference was apparent between the superficial fibrous region, which contained isolated foci of gap junctions (Fig 4B), and adjacent deeper zones of the lesion (Fig 4C), in which the foci were more numerous. This trend was apparent in six separate advanced atheromatous lesions examined.

In earlier atheromatous lesions (category 3), the connexin43 gap junction levels were markedly higher than in advanced atheroma, with a surface density of 36.9x10^-5 µm²/µm² section (±4.6 SEM). Fig 5 shows an example of such a lesion, together with a surrounding zone of intimal thickening. In regions of intimal thickening (category 2), the quantities of gap junctions were higher still at 152.3x10^-5 µm²/µm² (±19.1 SEM), a value approximately fourfold that of early atheroma. These features are illustrated in panels B through D of Fig 5. In diffusely thickened intimal regions, which contain multiple deposits of lipid, punctate gap-junctional labeling can be seen profusely distributed throughout the entire circumference of the thickened intima to the border of the small fibrolipid plaque (Fig 5B and 5C). By contrast, fewer gap junctions are apparent at the site of the plaque (Fig 5D). Although the size of the largest immunolabeled gap junctions in these lesions was similar to those in the advanced lesion category, small junctions (<1 µm) were markedly more abundant, particularly in the regions of intimal thickening.

View larger version (208K): [in this window] [in a new window]   Figure 5. Early atheroma and intimal thickening. The histological features (hematoxylin and eosin staining) of the lesion and adjacent areas of intimal thickening are illustrated in A, with three examples of confocal images from the regions indicated on the histological section as B, C, and D. Abundant punctate gap-junctional labeling is apparent compared with the images in Figs 2 through 4. A mixed population of large and small spots is seen, the latter predominating in regions of intimal thickening (panel C). Fewer, larger gap junctions are present in the developing cap of the small fibrolipid plaque (panel D), the base of which has a small, forming lipid pool (p in panel A). i indicates internal elastic lamina; L, lumen of vessel; Int, intima; and M, media. Bar=1 mm for panel A; bar=20 µm for B through D. Fig 6. Confocal image of undiseased segment of vessel. Low levels of gap-junctional labeling are apparent in the intima compared with Fig 5. L indicates lumen of vessel; M, media; Int, intima; and i, internal elastic lamina. Scale as in Fig 5B.

For comparison, an example of a confocal image of gap-junctional labeling in the normal arterial wall is illustrated in Fig 6. Sections of undiseased arteries showed significantly lower levels (P<.001) of labeling than those found in intimal thickening and early atheroma, with a value of 12.9x10^-5 µm² (±2.5 SEM) immunolabeled junctions per µm² section. The fluorescent spots were typically small and had a uniformly scattered distribution. By contrast, comparison of the quantity of gap-junctional labeling in undiseased vessels with that in advanced lesions revealed the latter to be significantly lower (P=.03).

That the immunolabeling observed throughout was specific was demonstrated by use of positive and negative controls. The heart sections stained in parallel always showed clear immunolabeling in the distinctive pattern of intercalated disks. No immunolabeling was observed (1) when the primary antibody was omitted and (2) when HH antibody was used in place of HJ.

In all samples, occasional labeling of the endothelium, identified by the position of the label in cells immediately adjacent to the lumen, was apparent. To establish which cell type within the arterial wall expresses the connexin43 gap junctions detected, double-labeling experiments were conducted to visualize gap junctions in combination with histochemically identified smooth muscle cells and macrophages. As demonstrated by the examples in Fig 7, gap-junctional immunolabeling was clearly associated with smooth muscle cells rather than with macrophages. The colocalization of gap junctions with smooth muscle cells is apparent when the two fluorescent images are presented in superimposed form (Fig 7B), although, owing to the relatively stronger fluorescence of the cell type marker compared with the small gap junction spots, some masking of the latter occurs. Clearer views are obtained when the gap junction labeling and histochemically identified smooth muscle cells in an identical area are presented as separate, side-by-side images (Fig 7C). Corresponding double-label experiments in which macrophages and gap junctions were localized revealed that macrophage-rich zones were not associated with gap junction label (Fig 7D), whereas zones containing fewer macrophages revealed some gap-junctional labeling predominantly in immunonegative regions between the marked cells (Fig 7E).

View larger version (2K): [in this window] [in a new window]   Figure 7. Double labeling of gap junctions (HJ polyclonal antibody) with (1) specific smooth muscle cell marker (HHF35 monoclonal antibody) and (2) specific macrophage marker (HAM56 monoclonal antibody). A, Survey view (hematoxylin and eosin section) of part of an early lesion showing the areas from which the confocal images in B through E were taken. (For complete view of this lesion, see Fig 5A). B, Double labeling of gap junctions (sharp green spots; arrowheads) and smooth muscle cells (red), with superimposition of the two images. Abundant gap junction labeling is associated with the presence of large numbers of smooth muscle cells. (For clarity of presentation, the colors of the original fluorescent probes have been reversed). C, Double labeling of gap junctions and smooth muscle cells.

View larger version (2K): [in this window] [in a new window]   Figure 7B. . (Continued, facing page). Here, the images of immunolabeled gap junctions (left) and smooth muscle cells (right) in an identical region of the lesion are presented side by side. This form of presentation makes it easier to see the extent of gap junction labeling in smooth muscle cell–rich regions than in superimposition images (B), in which the extent of smooth muscle cell fluorescence can mask the visibility of the much smaller punctate gap junctions. iel indicates internal elastic lamina. D and E, Double labeling (superimposed images) of macrophages (green) with gap junctions (orange spots marked by arrows). D is from a macrophage-rich region of the lesion; E is from an area in which macrophages are less abundant. No gap junction labeling is apparent in the image in D. Gap junction labeling in E is largely confined to areas that are immunonegative for macrophages. (Note that traces of nonpunctate dark red background are quite distinct from the sharp orange spots representing gap junctions). Bar=250 µm for A; bar=25 µm for B through E.

Further evidence that the gap junctions detected by immunolabeling were specifically associated with smooth muscle cells was obtained by thin-section electron microscopy (Fig 8). Gap junctions were seldom detected in advanced atherosclerotic lesions examined by thin sectioning, in line with their relative scarcity apparent by immunoconfocal microscopy. In earlier lesions, however, patches of small gap junctions were detected between groups of smooth muscle cells. The junctions were typically observed at points of interaction formed by finger-like processes extruded across regions of extracellular matrix (Fig 8A and 8B). No gap junctions were observed between macrophages.

View larger version (193K): [in this window] [in a new window]   Figure 8. Thin-section electron micrographs confirming abundance of gap junctions (arrows) between smooth muscle cells (Smc) from an early atheromatous lesion (A) and a region of intimal thickening identified as a fatty streak (B). Gap junctions typically occur at the ends of long cell processes that extend across the collagen (c) and other extracellular matrix components. Three separate gap junctions are present in the field shown in B. The inset in A (top left corner) shows a high-magnification view of the single gap junction present in the field, and C shows detail of a gap junction from an area close to that depicted in B. Both these high-magnification views reveal the typical pentalaminar structure characteristic of the gap junction. Bar=1 µm for A; bar=100 nm for inset; bar=5 µm for B; bar=100 nm for C.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study reveals that early stages of atherosclerosis are characterized by markedly increased expression of immunodetectable connexin43 gap junctions in the intima. As atherosclerosis progresses, however, the quantity of immunolabeled junctions declines, ultimately to levels below those of the undiseased vessel. With this decline, the distribution of the junctions becomes more patchy and the sizes of the individual junctions larger, as summarized diagrammatically in Fig 1.

Interactions between cells, particularly those involving extracellular signaling, play a central role in the pathogenesis of atherosclerosis.^{3 6} Direct intercellular communication via gap junctions has been implicated in the control of growth and differentiation in many systems,^{18 19 20 21 22} and these junctions have recently attracted attention in in vitro studies as possible modulators of function in the pathogenesis of arterial disease.^{23 24} Although the presence of gap junctions between neointimal smooth muscle cells has been noted in animal models of atherogenesis,^{45 46} the restricted sampling inherent in electron microscopy has prevented their detailed investigation, and no information has been available concerning the possible role of gap junctions in human arterial disease. By applying confocal microscopy, we obtained extensive high-definition views of immunofluorescently labeled gap junctions through the depths of 10-µm-thick tissue sections across human lesions. The relatively large volumes of tissue sampled by this technique reveal the overall amounts and patterns of connexin43 gap junction distribution and how these patterns change during lesion growth. Confocal immunodetection of gap junctions reveals the heterogeneity of junction distribution, a feature that would be difficult to detect with the restricted sampling attainable by thin-section electron microscopy.

The major gap-junctional protein expressed by smooth muscle cells is connexin43,^{27 47 48 49 50} and the antiserum used in the present study, directed against this connexin isoform, has previously been shown to bind specifically to smooth muscle cell gap junctions both in cultured cells²⁸ and in cells of the intact arterial wall.⁵¹ Expression of mRNA for an additional connexin isoform, connexin40, has been reported in cultured smooth muscle cells,⁵² and although this connexin has been localized in smooth muscle cells of small resistance vessels,⁵³ it does not appear to be present in significant quantities in the intact arterial wall.^{49 54} In contrast to smooth muscle cells, endothelial cells express connexin40 in both arterioles and large arteries.^{49 53} Endothelial cells also express connexin37⁵⁵ and, as confirmed here, connexin43.⁵⁶ In the present study, however, we observed only minor immunolabeling of connexin43 gap junctions between endothelial cells, any such immunolabeling attributable to this cell type being readily identifiable by its location at the luminal interface.

It has recently been reported that ATP-sensitive plasma membrane channels in macrophages consist of a protein that is homologous to connexin43⁵⁷ and that macrophage foam cells in the atherosclerotic plaque express connexin43 messenger RNA.²⁴ If the gap junction immunolabeling results were taken alone, it might be supposed that some of the observed signal was due to connexin43 expression by macrophages or macrophage/foam cells. This possibility was excluded by the negative correlation between gap junction immunolocalization and macrophages shown in our double labeling results. If ATP-sensitive membrane channels, homologous to connexons, were abundantly present and recognized by our HJ antibody, a diffuse signal might be predicted, reflecting a protein dispersed in the plasma membrane. However, no evidence for such a signal was apparent. Morphologically defined gap junctions involving macrophages or macrophage/foam cells were not observed by electron microscopy of the intact tissue. The significance of the reported expression of connexin43 transcripts in macrophages thus remains to be further evaluated.

That the observed connexin43 immunolabeling was confined to and representative of gap junction expression specifically by smooth muscle cells was demonstrated by (1) the double-labeling experiments (gap junction immunolabeling plus smooth muscle cell markers) and (2) the correlation found between the quantity of connexin43 immunolabeling detectable by confocal microscopy and the frequency of detection of ultrastructurally defined gap junctions between smooth muscle cells visible by electron microscopy at the different stages of lesion development.

We recently reported that expression of connexin43 gap junctions in cultured smooth muscle cells correlates with phenotype.²⁸ Transition from the contractile to the synthetic phenotype is accompanied by pronounced formation of connexin43 gap junctions in vitro. The markedly enhanced expression of connexin43 gap junctions observed in early lesions in the present study demonstrates that this correlation between the synthetic state and enhanced gap junction expression also applies in vivo. Smooth muscle cells are a normal component of the undiseased intimal wall of human coronary artery,⁴⁴ some of these cells being of the contractile phenotype and some synthetic. In early lesions, the cells that express gap junctions abundantly may in theory be derived either from preexisting intimal smooth muscle cells that have undergone transition from the contractile to the synthetic state or from cells that have migrated from the media.

Enhanced gap junction expression between smooth muscle cells thus appears early in the pathogenetic sequence, coincident with the initial accumulation of lipid and the onset of synthesis and accumulation of extracellular matrix components that account for the bulk growth of the lesion. Why and how the smooth muscle cells form gap junctions in such abundance at this stage is unclear, but it may be hypothesized that the complex actions of growth factors and cytokines involved in initiation of atherogenesis may act to enhance gap-junctional expression. For example, a close association between basic fibroblast growth factor and gap junctions has been reported in a number of systems.^{58 59 60} LDL (and specifically the apolipoprotein B moiety) has been shown to be a powerful stimulator of gap junction formation in cultured hepatoma cells,⁶¹ so the possibility exists that lipid accumulation at the earliest stages of atherogenesis itself serves as a direct stimulus to this initial phase of gap junction formation in smooth muscle cells of the arterial wall.

How might smooth muscle cell gap junctions contribute to progression of the atherosclerotic lesion? Smooth muscle cells in vitro have been shown, after several consecutive doublings, to adopt an irreversible synthetic phenotype,⁶² and direct communication between the cytoplasmic compartments of synthetic smooth muscle cells may contribute to the maintenance of this phenotype in the earlier stages of the disease. Gap-junctional communication would, in theory, permit passage of small regulatory molecules and ions between cells, either of which may influence phenotypic expression. Although the identity of such putative regulatory molecules is unknown, our results are consistent with a role for gap junctions in sustaining the activities of smooth muscle cells that result in growth of the plaque.

The subsequent decline in gap junction quantity with advancing stages of the disease parallels accumulation of extracellular matrix material. Gap junctions gradually become fewer, larger, and more sparsely scattered during this process. These changes may be explained in part by the increased separation between the cells brought about as they deposit matrix material around themselves. This increased separation appears to progressively reduce the areas over which adjacent plasma membranes can closely interact. Instead of multiple points of interaction over the cell surface, fewer sites of interaction are found, formed by long processes stretching across accumulating matrix. These circumstances dictate the presence of fewer junctions, and the observed increase in junction size may in part compensate for the enforced reduction in junction number. Ultimately, however, the quantity of extracellular matrix increases to the extent that, apart from isolated patches of smooth muscle cells, most of the cells in the lesion are unable to maintain any gap-junctional contact with their neighbors. By this time, however, the lesion is so advanced that gap-junctional communication is probably of little further significance to the genesis of the lesion or its fate.

	Acknowledgments

 
This study was supported by grants from the British Heart Foundation (No. PG/9316), Wellcome Trust, and The Royal Society.

Received April 28, 1995; accepted May 2, 1995.

	References

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