© 2001 American Heart Association, Inc.
Vascular Biology |
Regional Differentiation of Desmin, Connexin43, and Connexin45 Expression Patterns in Rat Aortic Smooth Muscle
From the National Heart and Lung Institute (Y-S.K., S.R.C., E.D., S.R., N.J.S), Imperial College, London, England, and The First Cardiovascular Division (Y-S.K.), Department of Internal Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan.
Correspondence to Prof N.J. Severs, National Heart and Lung Institute (Imperial College), Royal Brompton Hospital, Sydney Street, London SW3 6NP, England. E-mail n.severs{at}ic.ac.uk
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Abstract—The gap-junctional protein, connexin43, is differentially expressed in vascular smooth muscle cells (SMCs) according to phenotype. Previous studies suggest that desmin-negative SMCs are characterized by high levels of connexin43, whereas desmin-positive SMCs (of a more contractile phenotype) typically have low connexin43 levels. In this study, we examine systematically the inverse relationship between connexin43 and desmin in SMCs of defined regions of the rat aortic media and determine whether additional connexin isotypes are expressed and contribute to this relationship. Immunoconfocal microscopy demonstrated that (1) the inverse relationship between connexin43 and desmin expression holds true for the media of sequential aortic zones, with 1 exception, the ascending aorta, and (2) an additional vascular connexin, connexin45, is expressed by aortic SMCs. Examination of connexin43, connexin45, and desmin expression in sequential aortic zones reveals 3 SMC subpopulations. The first, predominating in the aortic arch and thoracic aorta, is desmin negative and contains high connexin43 levels; the second, predominating in the abdominal aorta and iliac artery, is desmin positive and contains low connexin43 levels; and the third, which is restricted to the ascending aorta, is desmin positive and expresses high connexin43 levels. Connexin45 levels are high in the ascending aorta but low in the other aortic segments. In para-aortic veins, a fourth SMC subpopulation appears, one that is desmin positive and contains connexin45 but not connexin43. These results demonstrate that a diversity of connexin expression patterns characterizes distinctive subpopulations of medial SMCs in situ with a potential to contribute to regional differentiation of vascular function.
Key Words: connexin43 • connexin45 • desmin • smooth muscle • aorta
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Introduction |
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The vascular system is regionally differentiated, reflecting adaptations to varying local demands and circulatory environments.1 In line with this heterogeneity, mature medial smooth muscle cells (SMCs) exhibit structural diversity according to their location in the vascular tree, manifested, for example, by differential expression of cytoskeletal proteins.2 SMC diversity has its origins in embryonic development and may contribute to distinct regional predilection for pathogenesis of specific vascular diseases.2 3 4 Apart from cytoskeletal proteins, recent evidence suggests that other proteins, in particular those of gap junctions, are also differentially expressed according to phenotypic diversification.5 6
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.7 The component proteins of gap-junctional channels, connexins, form a multigene family of conserved proteins, at least 16 members of which are expressed in mammalian cells. Work from in vitro expression systems demonstrates that gap-junctional channels composed of different connexins display distinct biophysical properties.7 Thus, there is considerable interest in identifying how specific expression patterns of different connexin isotypes contribute to the integration of cell and organ function in vivo.
In the cardiovascular system, the main connexin isotypes expressed are connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45).8 Whereas endothelial cells express multiple connexin isotypes (Cx37, Cx40, and Cx43),9 10 11 SMCs in vivo typically have a much simpler connexin expression profile. Cx43 is well established as the predominant (or sole) connexin of the medial SMCs of large arteries in vivo,8 11 although Cx40 is reportedly detectable in the SMCs of some smaller arteries and arterioles12 and is coexpressed with Cx45 and Cx43 in some types of nonvascular SMCs.13 14
Functionally, gap-junctional intercellular communication between vascular SMCs is implicated in the modulation of vasomotor tone and general circulatory homeostasis.15 16 Recent studies have further demonstrated levels of Cx43 gap junctions that are higher in vascular SMCs of the synthetic phenotype than in those of the contractile phenotype, suggesting a role related to synthesis and/or maintenance of extracellular matrix.8 17 18 19 20 However, differential Cx43 expression is not confined to phenotypic extremes but is also apparent between mature medial SMCs of more subtle phenotypic distinction. Cx43 is highly expressed in SMCs of the media of elastic arteries but occurs only at low or immunocytochemically undetectable levels in muscular arteries and arterioles21 ; in the human internal mammary artery, the SMCs expressing high Cx43 levels in the elastic medial regions are mainly desmin negative, whereas those with low Cx43 levels in the muscular medial regions are mainly desmin positive.5 Both SMC subpopulations are mature and express late differentiation markers, such as myosin heavy chain isoforms SM1 and SM2.22 Such an inverse relationship between Cx43 and desmin expression is also apparent in human coronary arteries5 and in cultured porcine aortic SMCs.6
From this background, we set out to examine systematically the consistency of the proposed inverse relationship between connexin43 and desmin and to identify whether other "cardiovascular" connexins are expressed in the SMCs of defined regions of the rat aorta. Previous studies in the rat suggest higher levels of Cx43 gap junctions in the proximal than in the distal aorta21 and more desmin-positive SMCs in the distal than in the proximal aorta,23 24 but the existing data are fragmentary. First, only Cx43 but no other major cardiovascular connexins were examined21 ; second, the initial part of the aorta, the ascending aorta, has not previously been investigated; and third, because these earlier studies were performed separately by different groups, no direct comparison between Cx43 and desmin profiles within the same study has previously been undertaken.
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Tissue Preparation
Samples of ascending aorta, aortic arch, thoracic aorta, abdominal aorta, and iliac arteries were obtained by rapid dissection from adult male Sprague-Dawley rats (300 to 450 g) killed by dislocation of the neck or CO2 asphyxiation. After they were rinsed with PBS containing heparin (10 U/mL), the samples were quickly cut into segments, frozen immediately in isopentane or in Cryo-M-Bed embedding compound (Bright) cooled with liquid nitrogen, and stored in liquid nitrogen or in a -70°C freezer. Ten-micrometer-thick cryosections of the vascular segments were cut transversely, mounted on poly-L-lysine–coated slides, and kept overnight in a -20°C freezer before immunolabeling. Animal work was conducted in accordance with the UK Animals (Scientific Procedures) Act, 1986.
Antibodies for Gap-Junctional Connexins,
SMC Differentiation Markers, and Endothelial Cell
Markers
We investigated by immunocytochemistry the 4 connexin
types now known to be the principal connexins expressed by
cardiovascular cells: Cx37, Cx40, Cx43, and Cx45. For
this purpose, we prepared a panel of polyclonal antibodies against
these connexins,8 of which the
following were used in the present study: for Cx37, Y16Y/R4
(dilution, 1:30) raised in rabbit; for Cx40, V15K/GP319 (dilution,
1:100) raised in guinea pig; and for Cx45, Q14E/GP42 (dilution, 1:100)
raised in guinea pig. All antibodies were affinity-purified,
characterized, and demonstrated to be isotype specific by Western blot
and immunofluorescence analysis of cells
transfected to express different connexin types and by immunogold
labeling at the electron-microscopic
level.5 9 10 25
For Cx43 single labeling, a mouse monoclonal anti-Cx43 antibody of
established specificity was used (Chemicon, dilution 1:1000). In
addition, to permit multiple labeling studies in which Cx43 could be
visualized simultaneously with other proteins that were
detected with the use of mouse and rabbit secondary antibodies, a new
anti-Cx43 polyclonal antibody was raised in chicken. This antibody,
designated S10C/CK84, was produced against a peptide sequence
corresponding to residues 314 to 322 of rat Cx43 (with an additional
C-terminal cysteine to facilitate coupling to the carrier protein) and
affinity-purified.10 25
This sequence has previously been reported for the production
of specific Cx43 antibodies in
rabbit.26
Commercially available mouse monoclonal antibodies were used
to detect the following proteins as SMC differentiation markers: desmin
(No. D1033, Sigma; 1:100), smooth muscle 
-actin (No. A-2547, Sigma;
1:1600), calponin (No. C-2687, Sigma; 1:10 000), and smooth muscle
myosin heavy chain isoforms (SM2, No. 7601, Seikagaku; 1:400).
Anti–von Willebrand factor (VWF) polyclonal antibody (Dako,
1:2500) raised in rabbit was used as a cell-specific marker for
vascular endothelium in double
labeling.11
The secondary antibody/detection systems used for immunoconfocal microscopy were donkey anti-mouse, anti–guinea pig, and anti-rabbit immunoglobulins conjugated to Cy3 (Chemicon, dilution 1:500), donkey anti-mouse immunoglobulin conjugated to Cy5 (Chemicon, dilution 1:500), donkey anti-rabbit immunoglobulin conjugated to FITC (Chemicon, dilution 1:50), and donkey anti-chicken immunoglobulin conjugated to Cy3 (Jackson, dilution 1:250). For immunoelectron microscopy, 10-nm gold/rabbit anti-chicken complexes (BioCell, 1:50) were used. Primary antibodies and secondary antibody/detection systems were diluted in 1% BSA in PBS (pH 7.4) before use.
Characterization of Anti-Cx43 (S10C/CK84)
Antibody
The specificity of the new anti-Cx43 (S10C/CK84)
polyclonal antibody raised in chicken was tested by
immunofluorescence labeling of HeLa cell
transfectants (kindly provided by Prof Dr Klaus Willecke, Institute
für Genetik, Bonn, Germany) and by immunogold thin-section electron
microscopy of rat
myocardium.10 25
Wild-type and Cx37-, Cx40-, Cx45-, and Cx43-transfected HeLa cells were
immersed in methanol at -20°C for 5 minutes and then in 1% BSA/PBS
at room temperature for 1 hour, incubated in the anti-Cx43 antibody
followed by donkey anti-chicken Cy3 at room temperature (1 hour each),
and examined by confocal
microscopy.10 25
For immunoelectron microscopy, specimens of rat ventricle were fixed
with 2% formaldehyde (freshly prepared from
paraformaldehyde) for 15 minutes and embedded in
Lowicryl K4M. Thin sections were labeled with the anti-Cx43 antibody
followed by 10-nm gold/rabbit anti-chicken complexes and examined with
a Philips EM301 electron
microscope.9 10 27
In addition to the use of connexin transfectants for confirmation of
antibody
specificity,5 9 10 25
routinely run negative controls included (1) omission of the primary
antibody and (2) a peptide inhibition test, in which the chicken
anti-Cx43 antibody was mixed with the S10C peptide (100 µg/mL, 30
minutes) before application to the cells and
sections.
Immunofluorescence Labeling
of Connexins and Differentiation/Cell-Type Markers
For immunofluorescence labeling
of aortic tissues, cryosections were fixed in methanol at -20°C for
5 minutes, washed with PBS, blocked with PBS/BSA at room temperature
for 60 minutes, and then incubated at room temperature in the selected
primary antibody or antibodies for 1 hour. After a washing with PBS,
incubation in the secondary antibody (or antibodies) followed (1 hour,
room temperature). The sections were then washed with PBS and mounted.
For single-labeling studies, guinea pig anti-Cx45 was detected with
donkey anti–guinea pig immunoglobulin conjugated to Cy3; for all mouse
monoclonal primary antibodies, detection was with donkey anti-mouse
immunoglobulin conjugated to Cy3. For double- and triple-labeling
studies, sections were treated sequentially with mixtures of primary
antibodies from different species and then with their corresponding
secondary antibodies conjugated to different fluorochromes
(except for the anti-Cx37
antibody, for which an overnight incubation was used before application
of other primary antibodies).
Negative controls included (1) omission of the primary antibody and (2)
for multiple labeling, using each primary antibody with matching and
nonmatching secondary antibodies. All secondary antibodies were
confirmed to be species specific to their individual primary
antibody.
Confocal Microscopy and Quantification
Confocal laser scanning microscopy of immunolabeled
sections was carried out with use of a Leica TCS 4D with an
argon/krypton laser (single-labeling studies) and a Leica TCS SP with
argon, krypton, and He/Ne lasers (multiple-labeling studies), both
equipped for the detection of FITC, Cy3, and Cy5 fluorescence.
The images were recorded by single- or sequential
dual/triple-channel scanning. Single optical sections and
projection views (for which sets of consecutive optical sections
were used) were examined.
To compare Cx43, Cx45, and desmin expression in different arterial regions, the immunoconfocal microscopic images of Cx43, Cx45, desmin, and ethidium bromide (20 µg/mL PBS for 1 to 5 minutes) labeling from 5 rats were used. For each immunolabeled component, 3 single optical images were acquired, with use of the x63 oil lens (with a theoretical focal depth of 0.5 µm), from 3 individual sections for each arterial region of each rat. From each image, a 900-µm2 sample area in the corresponding medial layer was randomly selected for quantification. The images were quantified by use of Scion Image analysis software (Scion Corp) as described previously.5 6 The number and individual areas of immunolabeled Cx43 and Cx45 gap-junctional spots, the total area of desmin signal, and the number of ethidium bromide–stained nuclei in each corresponding sample area were determined. These data provided indices of the mean area of gap-junctional spots, the numerical density of gap-junctional spots per cell, the mean area of desmin signal per cell, and the cellular density of each sample. It should be emphasized that the values given are not absolute values per cell but are comparative measures determined from a sample thickness of 0.5 µm; hence, they are referred to throughout as indices of the various parameters. The data from different arterial regions were presented as mean±SD and were compared statistically by the Wilcoxon rank sum test with statistical significance defined as P<0.05.
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Results |
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Characterization of Anti-Cx43 (S10C/CK84) Antibody
Immunofluorescence labeling of HeLa cell transfectants and immunoelectron microscopy of rat myocardium confirmed the specificity of the new affinity-purified anti-Cx43 (S10C/CK84) polyclonal antibody (Figure 1
). Whereas the wild-type and Cx37-, Cx40-, and
Cx45-transfected HeLa cells showed no positive signal for this antibody
(Figures 1A through 1D), prominent punctate labeling was
apparent at the cell borders between the Cx43-transfected HeLa cells
(Figure 1E). This labeling was inhibited by the corresponding
S10C peptide
(Figure 1F). In immunoelectron microscopy of
ventricular myocardium, gap junctions showing
the typical pentalaminar structure were specifically decorated by the
gold markers
(Figure 1G).
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Expression of Smooth Muscle Differentiation
Markers, Cx43, and Cx45 in the Rat Aorta and Iliac Artery
Of the SMC differentiation markers examined, whereas
smooth muscle -actin, calponin, and myosin heavy chain isoform SM2
were positively labeled in the majority of vascular SMCs in all
arterial regions studied, the labeling of desmin was
distinctly heterogeneous.
Figure 2 compares the labeling patterns of SM2 and desmin in
the different regions of the aorta and iliac arteries. For myosin heavy
chain isoform SM2, no detectable difference of the labeling intensity
was observed between different arterial segments
(Figures 2A, 2C, 2E, 2G, and 2I). In marked contrast, the
labeling pattern of desmin showed distinct differences according to
location along the aorta and the iliac artery
(Figures 2B, 2D, 2F, 2H, and 2J). In the ascending aorta
(Figure 2B), abdominal aorta
(Figure 2H), and iliac artery
(Figure 2J), the majority of the SMCs were positively labeled
with anti-desmin antibody, but in the aortic arch
(Figure 2D) and thoracic aorta
(Figure 2F), some of the cells were strongly labeled, whereas
others showed negative desmin labeling. The distinction between
desmin-positive and desmin-negative regions was clear-cut and without
intermediate grades. In the aortic arch
(Figure 2D), both cell groups were scattered across the
medial layers, without major segregation. The desmin-positive cells
were present at the luminal, middle, or adventitial side of the
media, without major preference. In the thoracic aorta
(Figure 2F), however, desmin-positive cells were
consistently more abundant at the luminal side of the vessel
than at the adventitial side.
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Cx43 and Cx45, but not Cx37 or Cx40, were detected in the
medial SMCs
(Figure 3). The distribution patterns of Cx43 and Cx45
punctate labeling in the medial layer varied according to the
arterial zone. The levels of Cx43 signals were high in the
ascending aorta
(Figure 3A), aortic arch
(Figure 3C), and thoracic aorta
(Figure 3E) but low in the abdominal aorta
(Figure 3G) and iliac artery
(Figure 3I). In the ascending aorta
(Figure 3A) and thoracic aorta
(Figure 3E), Cx43 labeling was more abundant at the
adventitial side of the vessel than at the luminal side, but such a
zonal difference was not apparent in the aortic arch
(Figure 3C), abdominal aorta
(Figure 3G), or iliac artery
(Figure 3I).
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For Cx45, the immunoconfocal labeling showed distinct
punctate signals in the medial regions of all vascular segments
examined
(Figures 3B
, 3D, 3F, 3H, and 3J). However, the sizes and
amounts of labeled Cx45 gap junctions varied in different
arterial segments. Cx45 spots were much larger and more
abundant in the ascending aorta
(Figure 3B) than in the other arterial segments.
No Cx45 signal was observed in the
endothelium.
Quantification of the number of vascular SMCs by counting
the cell nuclei (after ethidium bromide staining) showed that the cell
densities in the medial layer of the different arterial
zones were similar
(Figure 4A). Comparing the indices of the desmin signal area
per cell
(Figure 4B) and the numerical density
(Figure 4C) and mean area
(Figure 4D) of Cx43 gap-junctional spots per cell showed that
the relationship between Cx43 and desmin expression varied according to
arterial zone. An inverse relationship between Cx43 and
desmin expression was consistently apparent from the aortic
arch down to the iliac artery, with high Cx43 and low desmin levels at
the aortic arch and low Cx43 and high desmin levels at the iliac
artery. An exception was the ascending aorta, in which both proteins
were highly expressed. Quantification of the Cx45 signal showed that
the numerical densities of Cx45 gap junctions per cell were
significantly higher in the ascending aorta and decreased conspicuously
in the aortic arch and in the remainder of the distal artery
(P<0.0001)
(Figure 4E). The mean area of Cx45 gap junctions declined
progressively from proximal to distal arterial segments
(Figure 4F).
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Quantification demonstrated distinctive patterns of
desmin, Cx43, and Cx45 expression across the media in the different
arterial regions
(Figures 4G through 4I). In the ascending aorta, the area of
desmin signal per cell
(Figure 4G) and the numerical density of Cx43 and Cx45
gap-junctional spots per cell
(Figures 4H and 4I) were both significantly lower on the
luminal side than on the adventitial side
(P<0.0001 for Cx43, and
P<0.005 for Cx45). In the
thoracic aorta, the numerical density of Cx43 gap-junctional spots was
lower on the luminal side
(P<0.0001), but the area of
desmin signal per cell was higher
(P<0.0001). In the abdominal
aorta, both sides showed a similar tendency for low Cx43 and high
desmin levels (P>0.05). For
Cx45, no variation across the wall was apparent in the thoracic and
abdominal aorta
(P>0.05).
The relationships between Cx43, Cx45, and desmin were
further explored in multiple-labeling studies
(Figure 5). In the aortic arch
(Figure 5A) and thoracic aorta
(Figure 5B), the inverse expression pattern of Cx43 and
desmin was found to hold true down to the level of the individual cell,
as demonstrated by double labeling with the new anti-Cx43 antibody.
Although Cx43 labeling was abundant and prominent between
desmin-negative cells, it was much less frequent, and the spots were
smaller in size between desmin-positive cells
(Figure 5). For Cx45, the punctate labeling in the ascending
aorta was clearly seen between desmin-positive cells
(Figures 6A and 6B), and much of this signal was colocalized
with that for Cx43
(Figures 6C through 6F).
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Expression of Connexins in Small to
Medium-Sized Veins
Apart from its presence in the aorta and iliac artery,
Cx45 was also expressed abundantly in the small- to medium-sized veins
along the para-aortic regions, from the aortic root to distal iliac
arteries
(Figure 6G). Because internal elastic laminae in veins of
these sizes were not as obvious as those in large-sized veins and
arteries, the distinction between endothelium and
medial layers was difficult to define in the presence of elastic
autofluorescence, and the cellular localization of Cx45 cannot
be determined with certainty by single labeling. To overcome this
problem, Cx45 was colabeled with desmin and VWF for identifying SMCs
and endothelial cells, respectively. In the veins, the
majority of medial SMCs were desmin positive, and it became apparent by
triple labeling that Cx45 in the para-aortic veins was localized to
SMCs in the medial layers and not to the endothelium
(Figure 6H). Multiple-labeling studies for Cx40
(Figure 6I), Cx37
(Figure 6J), Cx43
(Figure 6K), and desmin with or without VWF demonstrated that
these 3 connexins were all expressed exclusively in the
endothelium but were never detected among the venous
SMCs.
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The present study demonstrates 2 key novel findings. First, the inverse relationship between Cx43 and desmin expression in medial SMCs holds true for sequential regions of the aorta down to the iliac artery, with 1 exception—the ascending aorta. Second, we show that in addition to Cx43, vascular SMCs in situ express a second connexin, Cx45. In the vascular system, although Cx45 transcript has previously been detected in a rat aortic SMC line (A7r5 cells),28 no other reports have previously demonstrated the presence of Cx45 gap junctions in vessel walls or in cultured vascular cells.
Systematic examination of Cx43, Cx45, and desmin expression
in the different zones of the rat aorta down to the iliac artery
reveals 3 SMC subpopulations. The first subpopulation, predominating in
the aortic arch and thoracic aorta, is desmin negative and contains
high Cx43 levels, whereas the second subpopulation, predominating in
abdominal aorta and iliac artery, is desmin positive and contains low
Cx43 levels. The previously identified inverse relationship between
Cx43 and desmin expression is thus maintained in these subpopulations.
The third subpopulation, which is restricted to the ascending aorta, is
different from those identified previously in that it is desmin
positive and also expresses high Cx43 levels. This subpopulation also
expresses high levels of Cx45, in contrast to the other regions, which
have lower levels of Cx45. The labeling for other SMC differentiation
makers, such as smooth muscle -actin (early-stage marker), calponin
(midstage marker), and myosin heavy chain isoform SM2 (late-stage
marker) was strongly positive in the medial layer of all
arterial zones examined, with no detectable regional
difference.22 In the
para-aortic small to medium-sized veins, however, a fourth vascular SMC
subpopulation, which is desmin positive and contains only Cx45 gap
junctions, is present. No other connexins are expressed at
detectable levels in any of the vascular SMC subpopulations; however,
Cx37, Cx40, and Cx43 are all expressed in the venous
endothelium.
The present results are in accord with the idea developed from our previous studies, ie, that Cx43 is expressed differentially in phenotypically distinct mature vascular SMCs and that desmin is the key differentiation marker to show any relationship with Cx43 expression.5 6 However, it is now apparent that the expression of an additional connexin, Cx45, has to be considered within this framework. The preferential distribution of 4 phenotypically distinct medial SMC subpopulations in different arterial and venous segments suggests that the regulation of Cx43, Cx45, and desmin expression is closely linked with regional diversity of the vascular system.
From previous studies, we suggested that high Cx43 levels in desmin-negative SMCs of elastic medial regions may be related to extracellular matrix production/maintenance, with low Cx43 levels in desmin-positive cells of muscular medial regions reflecting local regional control of blood flow.5 6 The present data extend this concept to the elastic medial regions of different anatomic locations within the aorta. In the rat, the SMCs of the abdominal aorta have higher contractile performance and express lower elastin levels than do those in the thoracic aorta.29 30 The general picture to emerge is that in the proximal elastic great arteries (aortic arch and thoracic aorta), the main SMC type consists of desmin-negative cells with high levels of Cx43, whereas in small- to medium-sized muscular arteries, the main cell type is desmin-positive cells with low levels of Cx43, with the abdominal aorta and iliac artery being of an intermediate transitional form with elastic media and desmin-positive/low-Cx43–expressing cells.
The occurrence of phenotypically distinct medial SMCs in
different locations may have its origin in embryonic
development.5 During
development, SMCs of different embryonic origin are distributed to
defined anatomic locations.31
The distribution boundary of SMCs of different embryonic origin in the
aorta appears to correspond to that demonstrated in the present
study between desmin-negative/high-Cx43 and desmin-positive/low-Cx43
SMCs. In the avian abdominal aorta and small muscular arteries, the
SMCs are known to be of mesodermal origin, whereas those of the aortic
arch and thoracic aorta are mainly derived from the ectodermal cardiac
neural crest.32 33
Functionally, although the participation of SMCs of ectodermal origin
is essential in the formation and organization of elastic laminae in
the great vessels,32 the SMCs
of mesodermal origin possess a significantly greater capacity for
contraction.33 In contrast to
other proximal elastic great arteries, the proximal end of the
ascending aorta, the coronary sinuses, does not (at least in
the chicken embryo) contain neural crest
cells.34 A recent report
suggests that in the rat, the medial SMCs of the proximal ascending
aorta may be transdifferentiated from embryonic
cardiomyocytes of the left ventricular outflow
tract,35 a cell lineage in
which Cx43 expression is high and positively correlated with desmin
expression.36 From the 12th
embryonic day, the cardiomyocytes in the distal truncal
part of the ventricular outflow tract, which is distal to
the emerging locations of the developing semilunar valves, begin to
shed their myocardial markers (- and ß-myosin heavy chain
isoforms) but retain SMC -actin and desmin, transforming into medial
SMCs of the proximal ascending
aorta.35
The discovery that Cx45 is expressed in SMCs at selected vascular sites extends these conclusions. With respect to the high Cx45 levels on SMCs of the ascending aorta, it is noteworthy that ventricular myocytes located at the adjacent proximal region, the subaortic area of the left ventricular outflow tract, similarly express high levels of Cx45.37 Gap junction channels composed of Cx45 have distinct biophysical properties compared with those made from Cx43 (eg, lower unitary conductance, stronger voltage dependence, and a lower anion-to-cation permeability ratio),38 39 potentially permitting distinctive intercellular communication properties according to the type of connexin expressed. Whether low quantities of Cx43 or Cx45 SMC gap junctions in the distal arteries are geared to the needs of delicate regional regulation of vasomotor tone and whether large quantities of Cx45 gap junctions in the venous desmin-positive SMCs are geared to the coordination of more extensive venous contraction (and hence, diastolic return) are yet to be established.
In conclusion, the identification of up to 4 SMC subpopulations in the rat aorta and para-aortic veins that differentially express Cx43, Cx45, and desmin provides a comprehensive picture of regional differentiation of connexin expression in phenotypically distinct medial SMCs of the vessel wall. Further studies are needed to explore the relationship of embryonic origin to vascular SMC phenotype and to determine the roles of SMC Cx43 and Cx45 gap-junctional intercellular communication in different regions of the vascular tree.
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Acknowledgments |
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This work was supported by the European Commission (QLRT-1999-00516). Yu-Shien Ko gratefully acknowledges personal support from an Overseas Biomedical Scholarship Award from the Chang Gung Memorial Hospital, Taipei, Taiwan.
Received May 2, 2000; accepted July 28, 2000.
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