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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1669-1680

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1669-1680.)
© 1999 American Heart Association, Inc.

Vascular Biology

Differential Expression of Connexin43 and Desmin Defines Two Subpopulations of Medial Smooth Muscle Cells in the Human Internal Mammary Artery

Yu-Shien Ko; Hung-I Yeh; Marcus Haw; Emmanuel Dupont; Riyaz Kaba; Gabriele Plenz; Horst Robenek; Nicholas J. Severs

From the National Heart and Lung Institute (Y.-S.K., E.D., N.J.S.), Imperial College School of Medicine, London; and the Department of Cardiovascular Surgery (M.H., R.K.), Harefield Hospital, Middlesex, UK; Mackay Memorial Hospital (H.-I.Y.), Taipei Medical College, Taipei, Taiwan; and the Institute for Arteriosclerosis Research (G.P., H.R.), University of Münster, Germany.

Correspondence to Prof Nicholas J. Severs, Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. E-mail n.severs{at}ic.ac.uk


*    Abstract
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Abstract—Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle {alpha}-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.


Key Words: gap junctions • connexin43 • desmin • smooth muscle cells • internal mammary artery


*    Introduction
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 *Introduction
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Transition of vascular smooth muscle cells (SMCs) from the differentiated contractile phenotype to a synthetic form that synthesizes large quantities of extracellular matrix materials is a key event in the pathogenesis of atherosclerosis.1 Interactions between SMCs and other cells and constituents of the arterial wall play a pivotal role in the modulation of SMC phenotype. These interactions involve a variety of signaling mechanisms, in particular those mediated by sensing specific components of the extracellular matrix and the action of growth factors.1 Recent evidence suggests that a further signaling mechanism, direct intercellular communication via gap junctions, may also participate in these interactions.2 3

Gap junctions are clusters of transmembrane channels that link the cytoplasmic compartments of neighboring cells, allowing direct exchange of ions, second messengers, and small signaling molecules. The gap-junctional channel is constructed from a pair of hemichannels termed connexons, each connexon being assembled from 6 connexin molecules. The connexins are a multigene family of conserved proteins, at least 13 members of which are expressed in mammalian cells.4 Four connexins, connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45), are reported to be expressed in the cardiovascular system.5 6 7 In the arterial wall, endothelial cells and SMCs have distinctive but overlapping connexin expression profiles.5 6 Although Cx40 and Cx37, and in some instances also Cx43, are coexpressed in the endothelium,8 SMCs in general show a simpler connexin profile, predominantly expressing Cx43.8 9 Cx43 is abundantly expressed in the media of elastic arteries, such as the aorta10 11 and carotid artery,3 but only at low, barely detectable or undetectable levels in muscular arteries, such as the coronary artery12 13 and arterioles.9 Some reports suggest that Cx40 and Cx45 may also be expressed in a few types of SMC.9 14 15 16

Gap-junctional intercellular communication between vascular cells is thought to contribute to general circulatory homeostasis, the modulation of vasomotor tone, and is implicated in the pathogenesis of hypertension and penile erection disorders.17 Recent evidence further suggests that increased Cx43-gap junction expression in arterial SMCs is intimately linked to transition to the synthetic phenotype, suggesting a possible role in regulation of extracellular matrix synthesis or other properties of the synthetic state cell. Cultured aortic SMCs express higher levels of immunodetectable Cx43 in the synthetic phenotype than in the contractile phenotype,18 and abundant Cx43-gap junctions are formed between intimal SMCs during the early stages of human coronary atherosclerosis2 and after balloon catheter injury in the rat carotid artery.3 During the development of the rat aorta, Cx43 expression is low in the media before birth but starts to increase postnatally, coincident with thickening of the media and deposition of elastic laminae.19 After reaching a peak at postnatal day 7, the levels of Cx43-gap junctions subsequently decrease to moderate levels on differentiation to the contractile phenotype as formation of elastic laminae is completed.19

In defining the relationship between Cx43 expression and SMC phenotype, it is important to bear in mind that multiple phenotypically distinct SMCs, each with different patterns of ultrastructural morphology, cytoskeletal protein and ion channel expression, electrical responses, capacity for extracellular matrix synthesis, migration, and growth, have been identified in adult arterial media and in culture. This has led to the concept that specific subpopulations of heterogeneous medial SMCs may be involved differentially in the cellular pathogenetic mechanisms of vascular diseases.20 21 Accordingly, the concept linking synthetic phenotype to abundant Cx43 expression and contractile phenotype to low Cx43 expression is likely to be overly simplistic, and the possibility exists that the pattern of connexin expression may differ within subpopulations of diverse SMCs in adult arterial media. An understanding of any such heterogeneity is required to assess more fully the relationship between gap junctions and properties associated with phenotypic state in the healthy and diseased human arterial wall.

To this end, the present study set out to explore the relationship between Cx43 expression and phenotypically distinct subtypes of SMC in the human internal mammary artery. The internal mammary artery is relatively resistant to atherosclerosis, and is the preferred choice of vessel for coronary artery bypass surgery,22 but our knowledge of the cellular basis of these properties has so far remained limited. The primary reason for selecting this vessel for study is that, in its distal region, it has an unusual histological pattern, ranging from elastic to elastomuscular to muscular,23 with SMCs of correspondingly distinctive morphology and intermediate filament expression patterns,24 thus providing an opportunity to compare the relationship between Cx43 expression and SMC heterogeneity within a single vessel. By characterizing the SMCs with respect to histological location, ultrastructural morphology, connexin expression (by immunohistochemistry and in situ hybridization), and differentiation marker expression (by immunohistochemistry), we show that Cx43 expression is high and desmin expression low in SMCs of elastic medial regions, whereas the converse characteristics apply to SMCs of muscular medial regions.


*    Methods
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Tissue Preparation
Specimens of distal left internal mammary arteries were obtained from 16 patients (15 males and 1 female; mean age, 61 years; range, 48 to 74 years) undergoing coronary artery bypass graft surgery because of severe coronary atherosclerosis. In addition, specimens of coronary artery from 2 patients (males; aged 40 and 52 years) undergoing heart transplantation for dilated cardiomyopathy were examined. The specimens were either fixed with 2.5% glutaraldehyde or rapidly frozen in isopentane cooled with liquid nitrogen. All work on human tissues was conducted according to institutional ethics committee policies. All immunolabeling and in situ hybridization experiments were conducted on cryosections of the rapidly frozen samples.

Thin-Section Transmission Electron Microscopy
The glutaraldehyde-fixed samples were postfixed with 2% osmium tetroxide and, after dehydration through an ethanol series, embedded in epoxy resin (Araldite CY212, Agar). Semithin sections were stained with toluidine blue and examined by light microscopy. Gold–silver thin sections were stained with uranyl acetate and lead citrate, and examined in a Philips EM301 or Hitachi H7000 electron microscope.

Immunofluorescence Labeling of Gap-Junctional Connexins, Vascular SMC Differentiation Markers, and Cell Type Markers
Four connexin types were investigated by immunocytochemistry, ie, Cx37, Cx40, Cx43, and Cx45. For immunofluorescence labeling of Cx43, we used a commercially available mouse anti-Cx43 monoclonal antibody directed against residues 252 to 270 of rat Cx43 (dilution, 1:1000; Chemicon). For immunofluorescence labeling of Cx37, Cx40, and Cx45, the primary antibodies used were a rabbit anti-Cx37 affinity-purified polyclonal antibody Y16Y(R4) (dilution, 1:30) raised against a peptide corresponding to residues 266 to 281 of rat Cx37, a guinea pig anti-Cx40 affinity-purified polyclonal antibody V15K(GP319) (dilution, 1:100) raised against a peptide corresponding to residues 256 to 270 of rat Cx40, and a guinea pig anti-Cx45 affinity-purified polyclonal antibody Q14E(GP42) (dilution, 1:100) raised against a peptide corresponding to residues 354 to 367 of human Cx45. The specificity of these antibodies has been demonstrated by western blotting and immunolabeling of HeLa cell transfectants, and by immunogold labeling (thin sections and/or freeze-fracture cytochemistry).7 25 Commercially available mouse monoclonal antibodies were used to detect the following proteins as SMC differentiation markers: desmin (1:100; Sigma, catalog No. D1033), vimentin (1:100; Boehringer Mannheim, catalog No. 1112457), smooth muscle {alpha}-actin (1:1600; Sigma, catalog No. A-2547), h-caldesmon (1:800; Sigma, catalog No. C-4562), calponin (1:10 000; Sigma, catalog No. C-2687), and smooth muscle myosin heavy chain isoforms, including SM1 (1:2000; Seikagaku, catalog No. 7599) and SM2 (1:400; Seikagaku, catalog No. 7601). Rabbit anti-Von Willebrand factor polyclonal antibody (1:2500; Dako) was used as a cell-specific marker to define the vascular endothelium in double labeling.8 Macrophages were detected by a mouse anti-CD68 monoclonal antibody (1:50; Dako, catalog No. M0876), and T lymphocytes by a mouse anti-CD3 monoclonal antibody (1:200; Chemicon, catalog No. MAB1731). The secondary antibody/detection systems used were donkey anti-mouse, anti-guinea pig, and anti-rabbit immunoglobulins conjugated to Cy3 and donkey anti-rabbit immunoglobulin conjugated to Cy5 (dilution, 1:500; Chemicon). Both primary antibodies and secondary antibody/detection systems were diluted in 0.5% BSA in PBS, pH 7.4, before use.

For immunofluorescence labeling, cryosections were fixed in methanol at -20°C for 5 minutes, washed with PBS, blocked with PBS/BSA for 30 minutes, and then incubated at room temperature in the selected primary antibody or antibodies for 1 hour (except for the anti-Cx37 antibody where an overnight incubation was used). After washing with PBS, incubation in the secondary antibody was for 1 hour at room temperature. The sections were then washed with PBS and mounted. For single-labeling studies, guinea pig anti-Cx40 and rabbit anti-Cx37 polyclonal antibodies were detected with donkey anti-guinea pig and anti-rabbit immunoglobulins conjugated to Cy3, respectively, whereas all mouse monoclonal primary antibodies were detected with donkey anti-mouse immunoglobulin conjugated to Cy3. For double-labeling studies, rabbit anti-Von Willebrand factor polyclonal antibody was applied mixed with either mouse anti-Cx43 or anti-desmin monoclonal antibody. The secondary antibodies, donkey anti-rabbit immunoglobulin conjugated to Cy5 and donkey anti-mouse immunoglobulin conjugated to Cy3, were also used as a mixture. Negative controls included (1) omission of the primary antibody, and (2) for double labeling, using each primary antibody with both matching and nonmatching secondary antibodies. All secondary antibodies were confirmed to be species specific to their individual primary antibody.

In Situ Hybridization
For in situ hybridization, human Cx37, Cx40, Cx43, and Cx45 cDNA were used. The production of Cx40, Cx43, and Cx45 clones and their characterization by northern blot have been reported previously.16 The human Cx37 (access No. M96789) cDNA was obtained by PCR amplification of human genomic DNA, using base 89 to 108 as upstream primer and base 1552 to 1571 as downstream primer.26 The PCR product was simultaneously cut with PstI and StuI endonuclease (Boehringer Mannheim), ligated into the vector pT7/T3 {alpha}18 (GibcoBRL) and the ligation mixture used to transform Escherichia coli. PCR product and recombinant plasmid were characterized and identified by restriction mapping. Northern blot analysis of RNA extracted from various tissues (all containing blood vessels) show a single band at {approx}1.8 kb as described26 (data not shown). After linearization of the plasmids for Cx37, Cx40, Cx43, and Cx45 with appropriate restriction enzymes, the digoxigenin–labeled sense and antisense RNA probes were generated by in vitro transcription, using a kit supplied by Promega.

In situ hybridization was performed after a procedure similar to that previously described.27 In brief, cryosections were fixed with 4% paraformaldehyde, dehydrated through an ethanol series, air-dried, and incubated at 52°C for 2 hours with prehybridization buffer (2x SSC, 50% formamide, 1 mg/mL BSA, 500 mg/mL tRNA, and 1 mg/mL denatured herring sperm DNA). Hybridization was done at 52°C overnight with digoxigenin-labeled probes (0.5 µg/mL in prehybridization buffer), and followed by RNase (Boehringer Mannheim) treatment (10 µg/mL for 10 minutes at room temperature) and washing with 4x SSC, 2x SSC, 1x SSC, and 0.1x SSC at 52°C. Sections were washed with TBS (10 mmol/L Tris, 150 mmol/L NaCl, pH 7.4) for 5 minutes, incubated with 0.5% blocking reagent (Boehringer Mannheim) in TBS for 1 hour and then with sheep anti-digoxigenin antibody (dilution, 1:500; Promega) in 0.5% blocking reagent/TBS for a further 1 hour. For detection, the sections labeled by Cx43 probes were immunostained with donkey anti-sheep immunoglobulin conjugated to Cy3 (dilution, 1:500; Chemicon) in 0.5% blocking reagent/TBS for 1 hour, and then were mounted for examination by confocal microscopy. The sections labeled with Cx37, Cx40, and Cx45 probes were immunostained with anti-digoxigenin–alkaline phosphatase, incubated with NBT (67.5 µg/mL) and BCIP (35 µg/mL) as substrates and counterstained with methylene green for the nuclei. Negative controls included (1) RNase A treatment before the prehybridization step, (2) hybridization with sense probes, and (3) omission of antisense probes.

Confocal Laser Scanning Microscopy
Immunolabeled sections from both immunofluorescence labeling of proteins and in situ hybridization were examined by confocal laser scanning microscopy, using a Leica TCS 4D, equipped with an argon/krypton laser and fitted with the appropriate filter blocks for detection of Cy3 and Cy5 fluorescence. A third channel corresponding to FITC fluorescence was used to record autofluorescent signal that mainly derives from elastic fibers.28 In addition, phase-contrast imaging was used, where appropriate, to enhance visualization of general tissue architecture. The images were taken by using single- or simultaneous dual-/triple-channel scanning; some were transformed into projection views by using sets of consecutive single optical sections. All specimens were examined within 24 hours of immunolabeling.

Quantification
To compare Cx43 and desmin expression in the media of internal mammary arteries, the immunoconfocal microscopic images of Cx43 and desmin labeling from 5 patients were used. For each immunolabeled component, three 400x images from 3 individual sections were randomly selected for each patient and, from each image, three 2304-µm2 sample areas in the media were randomly selected for quantification. For Cx43 labeling, the numbers and areas of immunolabeled Cx43 gap–junctional spots for each sample area were quantified by using PC Image analysis software (Foster Findlay Associates). A binary overlay was created automatically, and the threshold of the binary overlays was adjusted by comparing the original image to the optimal match of each spot. The value of the threshold setting was kept constant. For each sample area, the numerical density of gap-junctional spots (/103 µm2), gap-junctional size, and total gap-junctional area as a percentage of the total sample area were calculated. The total signal area in desmin labeling and the total elastic fibrillar autofluorescent area in each sample area were determined, and analyzed as percentage of labeled/fluorescence area in the total sample area.

To compare SMC densities in different medial areas, we used nuclear labeling by ethidium bromide (20 µg/mL PBS for 1 minute at room temperature). For each of 5 patients, 2 sections were randomly selected and a 160x confocal microscopic image (dual recording of FITC and Cy3 channels) was taken for each section. Two 14 884-µm2 sample areas were randomly selected from the medial area of each image. The number of nuclei in each sample area was counted, and the numerical density of SMC nuclei calculated. The elastic fibrillar autofluorescent area as a percentage of the total sample area was determined for each sample area as described above.

The relationship between autofluorescent elastic fibrillar area (elastic area, %), gap-junctional numerical density, gap-junctional size, total gap-junctional area, total desmin-positive area, and SMC density in the media of internal mammary arteries was evaluated by the Spearman rank correlation test.


*    Results
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Heterogeneity of Distal Internal Mammary Arteries
Toluidine blue staining of semithin sections and autofluorescence of elastic laminae in cryosections demonstrated a striking heterogeneity of structure in the distal internal mammary artery ranging from elastic to elastomuscular to muscular (Figure 1Down). This structural heterogeneity was typically apparent among samples from different patients and within each vessel, being readily demonstrated in sections of consecutive rings from the same arterial sample. Indeed even within the same transverse section, both elastic and muscular material was frequently observed interwoven in the medial layer. Concentric and eccentric intimal thickenings with amorphous and homogeneous autofluorescence were noted occasionally overlying both elastic and muscular medial regions. No advanced or complex atherosclerotic lesions were present in the samples examined.



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  		Figure 1. Heterogeneity of histological structure in the human distal internal mammary artery demonstrated by toluidine blue staining of semithin sections (A through C) and autofluorescence of elastic laminae in cryosections (D through F). The structure ranges from elastic (A and D) through elastomuscular (B and E) to muscular (C and F). Bar=10 µm (A through C) and 60 µm (D through F).

Vascular SMC Connexin Expression in Relation to Vessel Structure
The relationship of connexin expression by medial SMCs to the heterogeneous structure was studied by immunofluorescence labeling of Cx37, Cx40, Cx43, and Cx45 proteins and their corresponding transcripts examined by in situ hybridization.

Immunolabeling for Cx40 and Cx37 was positive in endothelium, but negative in medial and intimal SMCs of all samples of distal internal mammary arteries studied (data not shown). No Cx45 immunolabeling was observed in either the endothelium or the medial SMCs. Cx43 was the sole connexin detected in the media. In the media of elastic arterial segments, distinct Cx43 punctate labeling was prominent in the intervening spaces between autofluorescent elastic laminae (Figure 2ADown through 2C). By contrast, in muscular arterial segments, which typically showed scarce autofluorescent connective tissue, Cx43 labeling was sparse (Figure 2DDown through 2F). Elastomuscular arterial segments showed features between these extremes, with abundant punctate labeling of Cx43 in elastic regions containing autofluorescent thick laminae and fine fibrous networks, but only rare Cx43 labeling in muscular regions characterized by scarce autofluorescence. In regions of intimal thickening, distinct Cx43 punctate labeling was abundant and prominent (Figure 2GDown through 2I). The distribution pattern of connexin mRNA expression as determined by in situ hybridization was similar to that of protein expression (Figure 3Down). Cx43 mRNA expression was extensive in elastic medial regions and low in muscular medial regions (Figure 3ADown). Cx37 and Cx40 mRNA were detected in the endothelium but not in the media (Figure 3BDown and 3CDown), and Cx45 mRNA was not found in either endothelium or media (Figure 3DDown). Negative controls including those using connexin sense probes (Figure 3EDown through 3H) confirmed the specificity of in situ hybridization.



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  		Figure 2. Heterogeneous expression of connexin43 in the human distal internal mammary artery demonstrated by immunoconfocal microscopy. Phase-contrast images A, D, and G are taken from the same fields as the confocal microscopic images B, E, and H, respectively, to illustrate general histological features. C, F, and I are enlargements of the areas shown as boxes in B, E, and H, respectively. In all these images, the lumen (L) is at the top of the field. In the confocal microscopic images, the autofluorescent connective tissue appears as green fluorescence in the form of striated, network or amorphous structures, and connexin43-gap junctions appear as distinct red spots. Yellow signals arise either from autofluorescent lipofuscin bodies (arrows in E) or from the overlapping of green autofluorescent connective tissues and red signals in projections of optical section stacks (arrow in C). Connexin43-gap junction labeling is prominent and extensive in the elastic medial regions (B and C) but sparse in the muscular medial regions (E and arrowheads in F). In intimal thickenings, connexin43 labeling is abundant and prominent (H and I). IEL indicates internal elastic laminae; M, media; Int, intimal thickening. Dashed lines in G and H indicate position of the endothelium. Bar=20 µm (C, F, and I) and 100 µm (A, B, D, E, G, and H).



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  		Figure 3. Distribution of connexin transcripts in the human distal internal mammary artery demonstrated by in situ hybridization. Sections for connexin43 (Cx43) antisense and sense probes are labeled with Cy3 and viewed by immunoconfocal microscopy (A and E), and sections for other connexin probes are labeled by alkaline phosphatase substrate with methylene green counterstaining for nuclei (B through D and F through H). A shows that the extent of Cx43 mRNA is high in elastic medial regions (arrows) and low in muscular medial regions (arrowhead). B and C show that connexin37 (Cx37) and connexin40 (Cx40) mRNA are detected only in the endothelium (arrows), not in the media. Connexin45 (Cx45) mRNA is not detected either in the endothelium or in the media (D). Connexin sense probes are all negative (E through H), confirming specificity. L indicates lumen; IEL, internal elastic laminae; M, media. Bar=50 µm.

The consistent pattern to emerge from these observations was that SMC Cx43 expression was high in elastic medial regions comprising prominent autofluorescent fibrous networks, but markedly lower in muscular medial regions with scarce autofluorescent connective tissue. High levels of Cx43 expression also consistently occurred in intimal thickenings.

Vascular SMC Phenotype in Relation to Cx43 Expression
To determine whether the SMCs that express different levels of Cx43 are phenotypically distinct, ultrastructural examination and immunofluorescence labeling using a panel of SMC differentiation markers were performed.

Thin-section electron microscopy revealed that although SMCs of both elastic and muscular medial regions both showed morphological features typical of the contractile phenotype such as abundant, well-organized myofilaments, distinct differences were nevertheless apparent between them (Figure 4Down). Whereas SMCs of elastic medial regions were irregular in shape with numerous processes, surrounded by large areas of connective tissue (Figure 4ADown), those of muscular medial regions were of spindle shape with few processes, and were closely approximated to one another with less intervening connective tissue (Figure 4BDown). Synthetic organelles in SMCs of the elastic medial regions were more conspicuous than those of the muscular medial regions (Figure 4ADown and 4BDown). Gap junctions between SMCs of elastic medial regions were relatively frequently detected (Figure 4CDown), but few gap junctions could be found between SMCs of muscular medial regions. In intimal thickenings, the SMCs were small, irregular, contained variable quantities of secretory organelles, and appeared loosely embedded in connective tissue; gap junctions were also frequently demonstrated between these cells (Figure 4DDown).



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  		Figure 4. Thin-section electron micrographs showing heterogeneous ultrastructure of smooth muscle cells (SMCs) in the human distal internal mammary artery. In elastic medial regions (A), the SMCs are irregular, are rich in synthetic organelles (so), and are surrounded by extensive connective tissue, especially elastic laminae (el). In muscular medial regions (B), the SMCs are typically spindle shaped, with fewer synthetic organelles and less connective tissue. Typical gap junctions (gj) are frequent between SMCs in elastic medial regions (C) and intimal thickenings (D). The insets show enlarged views from these regions (indicated by arrows) in which the diagnostic pentalaminar structure of the gap junction is clearly seen. smc indicates smooth muscle cells; l, lipid; p, cell process of intimal SMC. Bar=1 µm (A through C) and 0.5 µm (D).

The expression of desmin, vimentin, smooth muscle {alpha}-actin, h-caldesmon, calponin, and myosin heavy chains (SM1 and SM2) was evaluated by immunofluorescence labeling (Figures 5Down and 6Down). Distinct desmin labeling patterns were observed according to vessel structure (Figure 5ADown and 5BDown). Whereas desmin labeling was prominent and extensive in muscular medial regions (Figure 5ADown), desmin-positive SMCs were scarce in elastic medial regions (Figure 5BDown). The labeling of vimentin, smooth muscle {alpha}-actin, h-caldesmon, calponin, SM1, and SM2 was positive in most medial SMCs examined, with no detectable differences between elastic and muscular medial regions (Figure 5CDown through 5H). In the intimal thickenings, although desmin is rarely detected (Figure 6ADown and 6BDown), vimentin, smooth muscle {alpha}-actin, h-caldesmon, calponin, SM1, and SM2 were positive in most SMCs (Figure 6CDown through 6H). Labeling for macrophages by anti-CD68 antibody and for T lymphocytes by anti-CD3 antibody was consistently negative in intimal thickenings.



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  		Figure 5. Immunoconfocal images of differentiation markers in the medial smooth muscle cells of internal mammary artery. Desmin labeling is high in muscular medial regions (A) but low in elastic medial regions (B). Vimentin, smooth muscle {alpha}-actin, h-caldesmon, calponin, and myosin heavy chain isoforms (SM1 and SM2) are all prominently expressed by most medial smooth muscle cells, with no detectable differences between muscular and elastic regions (C through H). L indicates lumen; IEL, internal elastic laminae; M, media. Bar=50 µm.



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  		Figure 6. Immunoconfocal images of smooth muscle cell differentiation markers in the intimal thickenings of internal mammary artery. Phase-contrast image A is taken from the same field as the confocal microscopic image in B, to illustrate general histological structure. Whereas desmin is rarely detected in the intimal thickenings (B), vimentin, smooth muscle {alpha}-actin, h-caldesmon, calponin, and myosin heavy chain isoforms (SM1 and SM2) are expressed by most smooth muscle cells in the intimal thickenings (C through H). L indicates lumen; Int, intimal thickening; IEL, internal elastic laminae; M, media. Bar=30 µm.

Thus, among the SMC differentiation markers used in this study, desmin was the only one found to be differentially expressed in elastic versus muscular medial regions. Moreover, the expression and distribution pattern of desmin in the different SMC types was inversely related to that of Cx43. Figure 7Down illustrates this relationship by comparing serial sections showing similar areas of intimal thickening, elastic and muscular medial regions labeled with Cx43 and desmin, respectively.



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  		Figure 7. Immunoconfocal images of serial sections demonstrating the inverse pattern of connexin43 (A) and desmin (B) expression in internal mammary artery. Smooth muscle cells in intimal thickenings (Int) and elastic medial regions (El) express high levels of connexin43 (red spots in A) but low levels of desmin, whereas those of muscular medial regions (Muscu) express low levels of connexin43 but high levels of desmin (red patches in B). Von Willebrand factor (VWF) is colabeled and shown in blue to define the endothelium (Endo). L indicates lumen; IEL, internal elastic laminae. Bar=60 µm.

To determine whether a similar inverse relationship of Cx43 and desmin expression held for other vessels, human coronary arteries were also examined. Desmin expression in the coronary artery was found to show distinct zonal variation, being highest in the outer medial zone, lower in the inner medial zone, much lower in the outer intimal zone, and almost undetectable in the inner intimal zone (Figure 8ADown and 8BDown). The magnitude of Cx43 expression showed a clear inverse relationship to that of desmin in these zones (Figure 8CDown).



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  		Figure 8. Immunoconfocal microscopy and corresponding phase-contrast imaging of serial sections of the human coronary artery demonstrating that an inverse relationship between desmin and connexin43 expression is also apparent in the intimal and medial layers of the vessel. Phase-contrast image A is taken from a corresponding field to the confocal microscopic images B and C to illustrate general histological structure. (a) through (d) are enlarged areas indicated by boxes in C, to show the details of connexin43 labeling. Desmin labeling is highest in the outer medial zone and undetectable in the inner intimal zone, with the inner medial and outer intimal zones showing intermediate labeling levels (B). The distribution of connexin43 labeling clearly shows the reverse pattern (C). The connexin43 gap–junctional spots are huge and at high density in the inner intimal zone (a), but smaller and much less frequent toward the outer adventitial side (b and c). In the outer medial zone, the spots are tiny and scarce (arrow in d). L indicates lumen; Int, intimal thickening; IEL, internal elastic laminae; M, media; EEL, external elastic laminae. Bar=120 µm (A through C) and 24 µm (a through d).

Statistical Correlation of Desmin and Cx43 Expression With Elastic Autofluorescence in the Medial Layers of the Internal Mammary Artery
The observations were confirmed by quantitative analysis (Figure 9Down). The quantity of elastic autofluorescence showed a negative correlation with extent of desmin labeling (r=-0.6943, P<0.0001, n=45), and a positive correlation with gap-junctional numerical density (r=0.7402, P<0.0001, n=45), total gap-junctional area (r=0.7227, P<0.0001, n=45), and gap-junctional size (r=0.4422, P=0.0024, n=45). The SMC densities evaluated by ethidium bromide labeling were similar in medial zones showing different degrees of elastic autofluorescence (r=-0.02, P=0.9, n=30), demonstrating that the results were not affected by differences in cell number/unit area.



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  		Figure 9. Statistical analysis showing that the quantity of elastic autofluorescence (elastic area, %) in the media of human distal internal mammary artery is negatively correlated with the extent of desmin labeling (A), but positively correlated with gap-junctional (GJ) numerical density (B), total gap-junctional area (C), and individual gap-junctional size (D).


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
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The principal novel finding of the present study is that inverse patterns of Cx43 and desmin expression define 2 distinctive subpopulations of SMCs in elastic and muscular medial regions of the distal internal mammary artery. SMCs in the muscular medial regions express high levels of desmin but low levels of Cx43, whereas those of elastic medial regions express low levels of desmin but high levels of Cx43. Morphologically, the former cells are typically spindle shaped with rare synthetic organelles, whereas the latter are irregular in shape with more prominent synthetic organelles. The coexistence of these distinctive SMC subpopulations within the same vessel appears to be directly related to the unique structure of the distal portion of the internal mammary artery. The internal mammary artery is the only medium-sized artery to possess an elastic component like that of elastic arteries, in addition to typical muscular features.23 This complex nature may have its origin in embryological development, as the internal mammary artery forms from anastomoses of thoracic intersegmental arteries that develop directly from the dorsal aorta.29 Apart from the medial SMCs, zones of intimal thickening were also identified occasionally, and the SMCs in these zones more closely resemble those of the elastic medial regions than those of the muscular medial regions with respect to patterns of Cx43 and desmin expression.

Our observation that Cx43 protein expression detected by immunolabeling and Cx43 mRNA expression detected by in situ hybridization showed a similar distribution pattern in the media of the internal mammary artery suggests regulation of SMC Cx43 expression at the transcriptional level. As Cx43 is only 1 of 13 connexin types identified in mammalian cells, the possibility existed that other connexins might be expressed (and, hence, gap junctions formed) in the Cx43-deficient SMC population. Although a second connexin, Cx40, has been reported in the SMCs of some arterioles and in the A7r5 aortic SMC line,9 30 we found no evidence for Cx40 labeling in elastic or muscular medial regions of the internal mammary artery. We similarly found no evidence for Cx37 or Cx45 expression in the SMCs examined. Thus, our present findings are in accord with the view that Cx43 is the sole detectable connexin expressed in vivo by SMCs of large- and medium-sized arteries.5 6

Among the markers of SMC differentiation used in this study, smooth muscle {alpha}-actin is the earliest to be expressed during vascular development.31 Calponin and h-caldesmon are considered intermediate-stage markers of SMC maturation, and myosin heavy chain isoforms SM1 and SM2 are considered to be late-stage markers.31 Various expression patterns of smooth muscle {alpha}-actin, calponin, h-caldesmon, and myosin heavy chain isoforms have been reported in a range of vessels from different species.31 However, our present finding that in the human internal mammary artery smooth muscle {alpha}-actin, calponin, h-caldesmon, SM1, and SM2 were all typically highly expressed, and that, ultrastructurally, myofilaments were abundant, indicates that, despite some ultrastructural differences, the SMCs of both subpopulations may be classified as variants of a differentiated contractile phenotype.

In marked contrast to these markers, differential expression of the intermediate filament protein, desmin, defines a prominent phenotypic difference between these 2 SMC variants. Desmin is closely linked to the contractile function of muscle tissue,32 and its expression in vascular SMCs is characteristically associated with the contractile phenotype rather than with the synthetic phenotype. At the embryonic stage, desmin is rarely detected in the aorta and is present only in a small proportion of muscular arteries.33 34 35 A preferential association of desmin-positive SMCs with muscular arteries and of desmin-poor SMCs with elastic arteries becomes apparent as desmin expression rises after birth and with age.33 36 For example, low SMC desmin content has been reported in embryonic and neonatal pulmonary arteries during active elastogenesis,37 with the proportion of desmin-positive cells increasing to intermediate levels in adult elastic arteries and high levels in adult muscular arteries and arterioles.38 39 40 41 Thus, although both SMC subpopulations were identified as a differentiated contractile phenotype, that with high desmin levels may be considered as showing more highly developed contractile features than that expressing low desmin levels.

Our finding of distinctly different levels of desmin expression in subgroups of SMCs that appear to show similar levels of vimentin is noteworthy, given that the expression of these 2 intermediate filament proteins in vascular SMCs generally shows an inverse relationship (ie, cells more usually show either high desmin with low vimentin levels or low desmin with high vimentin levels). For example, in experimental animals, vimentin is typically highly expressed in elastic arteries (eg, aorta) but found in low levels in muscular arteries (eg, femoral artery), with desmin showing the reverse pattern.38 39 40 However, in keeping with our current findings in the human internal mammary artery, vimentin labeling is reported to be uniformly positive in intimal and medial SMCs of adult human aorta and muscular arteries.41

As SMC differentiation progresses within a wide phenotypic spectrum,31 our findings suggest that differences in Cx43 expression are not only linked to extremes of SMC phenotype, but also occur between cells of more subtle phenotypic difference, in this case differentiated SMCs with distinctly different desmin expression levels. It should, however, also be noted that distinct subpopulations of SMCs may originate from separate embryological cell lineages. For example, recent evidence suggests that SMCs derived from ectodermal cardiac neural crest, rather than those derived from mesoderm, play the central role in vasculogenesis and elastogenesis of the outflow tract.35 Cx43 is highly expressed by the neural crest cell lineage and is associated with the development of proximal great vessels.42

The inverse relationship between Cx43 and desmin expression in the internal mammary artery is not only a feature of whole groups of cells; data from preliminary double-label experiments on cultured cells indicate that the relationship applies at the level of the individual cell (Y.-S.K.) (unpublished data, 1998). This relationship is different from that in cardiac myocytes and other visceral SMCs (eg, those of the uterus). During the differentiation of mouse cardiac myocytes, expression of Cx43 is positively correlated with that of desmin.43 In a similar manner, in myometrial SMCs, both desmin44 and Cx4316 45 are upregulated in late pregnancy. In both these cases, the presence of high levels of desmin reflects active contractile properties of the cells with the concomitant abundance of Cx43 linked to coordination of electromechanical activity. The fact that this relationship does not hold for arterial SMCs not only suggests that the Cx43/desmin relationship is organ or tissue specific, but that the presence of gap junctions in arterial SMCs may be primarily associated with function(s) other than coordination of contraction.

The present findings refine the view suggested from previous studies2 3 18 19 that high levels of Cx43 expression are associated with SMCs of a less contractile and more synthetic state, typically in an environment rich in extracellular matrix. These observations suggest that Cx43 gap–junctional intercellular communication may somehow be linked to the ability of SMCs to coordinate synthesis of extracellular matrix components, maintain the functional integrity of elastic media and/or regulate the repair and formation of the intima in vascular injury and disease. If this speculation is correct, then the observation that Cx43 expression is high in areas both of abundant elastic fibers (such as the elastic media) and of collagen (such as the intima) might suggest that Cx43 gap–junctional intercellular communication is related more to synthesis/presence of extracellular matrix, in general, than to the specific type of product. In contrast, it may be that the presence of abundant extracellular matrix or other properties of the synthetic (or less contractile) phenotype are the stimulus for increasing Cx43 expression. Further functional studies are needed to explore the possible relationship between Cx43 gap–junctional intercellular communication and synthesis, deposition, and removal of specific extracellular matrix components in physiological and pathological states.


*    Acknowledgments
 
This work was supported by the British Council and German Academic Exchange Service ARC Program (Project 815), and in part by grants from the Wellcome Trust (046218/Z/95) and the British Heart Foundation (grant PG/97175). Dr Yu-Shien Ko is a cardiologist from Chang Gung Memorial Hospital, Taipei, Taiwan, and gratefully acknowledges personal support from its Overseas Biomedical Scholarship Award. We thank Stephen Rothery for his expertise with confocal microscopy and figure preparation.

Received November 27, 1998; accepted December 17, 1998.


*    References
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