© 2000 American Heart Association, Inc.
Vascular Biology |
Multiple Connexin Expression in Regenerating Arterial Endothelial Gap Junctions
From Mackay Memorial Hospital (H.-I.Y., Y.-J.L., H.-M.C., C.-H.T.), Taipei Medical College, and the First Cardiovascular Division (Y.-S.K.), Department of Internal Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan; and the National Heart and Lung Institute (Y.-S.K., N.J.S.), Imperial College School of Medicine, London, UK.
Correspondence to Cheng-Ho Tsai, Cardiac Medicine, Mackay Memorial Hospital, 92, Sec 2, North Chung San Road, Taipei 10449, Taiwan. E-mail cht7678{at}ms2.mmh.org.tw
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Abstract |
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Abstract—Endothelial cells form gap junctions that, according to vessel type, may be composed of up to 3 types of connexin, connexin37, connexin40, and connexin43. Although changes in connexin expression have been linked to growth and injury in cultured endothelial cells, information on connexin expression in regenerating endothelium in situ is lacking. We investigated gap junction distribution and expression of all 3 endothelial connexins during healing in rat carotid artery after denudation injury. En face viewing of the vascular luminal surface by means of immunoconfocal microscopy was used to examine the spatial and temporal expression pattern of the endothelial connexins. Gap junction spots labeled by specific antisera against connexin37, connexin40, and connexin43 were quantified 7, 14, and 28 days after injury, and the relations among the connexins were examined by using colocalization analysis. Complementary electron microscopy was also conducted. After injury, the regenerating endothelium initially expressed small, sparse gap junctions, the numbers of which progressively increased to values equivalent to those of controls. Although connexin40 gap-junctional spot size and area returned to uninjured levels by 28 days after injury, connexin37 and connexin43 spot size and area exceeded those of the uninjured artery (P<0.05). Double-label analysis showed that even though colocalization of connexins to the same gap-junctional spot is a common feature, the extent of colocalization was time dependent (>80% in the intact artery at postinjury day 28 and <70% at postinjury days 7 and 14, P<0.01). We conclude that distinct alterations in expression of the 3 connexins are associated with regeneration of the arterial endothelium in situ, implying different intercellular communication requirements during the various phases of the healing process.
Key Words: gap junctions • connexin • endothelial cells • regeneration
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Introduction |
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Vascular endothelial cells interconnect to form a single layer covering the luminal surface of the blood vessel. This anatomic location makes the endothelium a primary target for injury by blood-borne atherogenic factors, such as those associated with hyperlipidemia, diabetes, hypertension, and smoking.1 2 In the clinical course of atherosclerosis, damage to the endothelium may arise not only from the disease itself but also from angioplasty.3 Depending on the severity of injury, endothelial cells manifest dysfunction or desquamation followed by regeneration, the ability of which is critical to inhibition of proatherogenic processes, such as phenotypic transformation of the underlying smooth muscle cells.4 5 Regulation of endothelial cell regeneration is thought to involve a range of cell-cell signaling processes, including those mediated by gap junctions.6 7
Gap junctions are clusters of protein channels that link the cytoplasmic compartments of neighboring cells, allowing direct exchange of signaling molecules (<1000 Da) and ions.8 9 The component proteins of these channels, connexins, belong to a multigene family. Of the 16 members identified in mammalian cells, connexin37 (Cx37), Cx40, and Cx43 are known to be expressed in various levels in different types of endothelial cells.10 11 12 13 14 Studies in in vitro expression systems indicate that the properties of gap-junctional channels are determined by their connexin makeup and that the function of channels made of 2 connexins differs from that of channels made of either 1.15 16 17 Thus, endothelial cell gap junctions, containing up to 3 connexins, possess considerable potential for modulation of specific intercellular communication properties according to functional need.
Gap junctions are implicated in a variety of endothelial activities, notably maintenance of monolayer topology, coordination of vasomotor responses, and regulation of angiogenesis, endothelial growth, and senescence.6 18 19 20 21 The specific pattern of connexin expression may also be differentially affected by physical and chemical factors, such as mechanical load, blood sugar level, growth factors, and cytokines.21 22 23 24 25 However, current knowledge about the role of each connexin in the endothelial cell is limited, and most studies have been limited to examination of 1 or 2 connexins. Previous studies of regenerating vascular endothelium in animal models have been confined to morphometric measurement of the size and number of gap junctions, and corresponding in vitro studies have been confined to Cx43.19 22 26
Our previous work suggested that gap junctions are upregulated on phenotypic transformation of vascular smooth muscle cells after injury.27 28 However, the role of individual connexins and their interrelationship in the overlying endothelium in this process is not known. Therefore, we investigated the spatial and temporal expression patterns of endothelial gap junctions and connexins during vascular healing in rat carotid artery after denudation injury. Our previous work with this model showed that Cx37 and Cx40 are present only in the endothelial cells, whereas Cx43 is expressed in both the endothelium and underlying smooth muscle.28
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Methods |
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Samples and Tissue Processing
Adult male Sprague-Dawley rats (300 to 400 g) were anesthetized with sodium pentobarbital (50 mg/kg IP), and the left common carotid artery was denuded of endothelium by means of cephalic-to-caudal passage of polyethylene (PE50) tubing (Clay Adams), which was done 3 times. At least 3 cm of the tubing was introduced into the artery to ensure that the tip reached the aorta–carotid artery junction. After the tubing was removed, the artery puncture site was sealed with tissue adhesive (Vetbond, 3M Co). The uninjured right carotid artery served as a control.
Immediately after the operation, 4 animals were injected with 1 mg/kg of Evans blue dye (Sigma) in PBS through the femoral vein and were perfusion fixed for assessment of the extent of injury. Twenty-one animals were killed (without administration of Evans blue) 7, 14, and 28 days after injury (7 animals per group) for immunoconfocal study of connexins. These animals were anesthetized (with pentobarbital administered intraperitoneally) and perfused retrogradely, by means of a catheter placed in the abdominal aorta, with heparinized PBS (10 U/mL) followed by phosphate-buffered 2% paraformaldehyde (pH 7.4) for 10 minutes. The proximal portions of the carotid arteries and the connecting aortic arch were dissected and stored under liquid nitrogen before immunolabeling. Selected arterial samples were prepared for thin-section electron microscopy by using standard procedures.28 The work was conducted in accordance with the Republic of China Animal Protection Law (Scientific Application of Animals) of 1998.
Immunofluorescence Labeling of Connexins and
Endothelial Cell Identification
Anti-Connexin Antibodies and Endothelial Cell
Marker
Three principal antibodies were used for
immunofluorescent detection of Cx37, Cx40, and Cx43. The Cx37
polyclonal antiserum, designated Y16Y(R3), was raised in rabbits
against a synthetic peptide corresponding to residues 266 to 281 of the
cytoplasmic C-terminal tail of rat Cx37.The Cx40 polyclonal antiserum,
designated V15K(GP319), was produced in guinea pigs against a synthetic
peptide corresponding to residues 256 to 270 of the cytoplasmic
C-terminal tail of rat Cx40. These two antisera were affinity purified
against the appropriate peptide coupled to an activated
chromatography matrix. The connexin isotype specificity
of each purified antibody was confirmed by
immunofluorescence and Western blot
analysis of cells transfected to express different connexins.
This approach demonstrates that neither antibody cross-reacts with
other connexins.13 More comprehensive characterization was
performed by using immunogold labeling and electron
microscopy.13 For Cx43, a mouse monoclonal antibody of
widely established reliability was purchased from Chemicon.
Endothelial cells were identified using rabbit
anti-human von Willebrand factor polyclonal antiserum
(Dako).
Secondary Antibody and Detection Systems
Donkey anti-rabbit, anti–guinea pig, and anti-mouse
immunoglobulin conjugated to CY3 or CY5 (Chemicon) were used to
visualize immunolabeled connexins. For single labeling of individual
connexins, CY3-conjugated antibodies were used. For double labeling of
2 connexins, 1 CY3-conjugated antibody and 1 CY5-conjugated were used
in combination. In selected experiments in which Cx43 was visualized
with CY3, simultaneous endothelial cell
marking was performed using anti–von Willebrand factor
detected with donkey anti-rabbit-CY5.
Immunolabeling of Connexins
For single labeling of 1 connexin, samples were cut into rings,
blocked in 0.5% BSA (15 minutes), and incubated with anti-Cx37 (1:60),
anti-Cx40 (1:100), or anti-Cx43 (1:500) at 37°C for 2
hours.13 Samples were then treated with CY3-conjugated
secondary antibody (1:500, room temperature, 1 hour). In experiments in
which 2 connexins were simultaneously localized in the same
samples, the samples were incubated in a mixture of anti-Cx37 plus
anti-Cx40, anti-Cx37 plus anti-Cx43, or anti-Cx40 plus anti-Cx43,
followed by a mixture of the 2 corresponding species-specific secondary
antibodies (CY3 and CY5; 1:500). When simultaneous marking
of endothelial cells was performed, single-labeled
samples were incubated with anti–von Willebrand factor
(1:500). Finally, arterial rings were cut open and mounted
for en face viewing of endothelial cells. All
experiments included sections of rat heart as positive
controls11 ; primary antibody was omitted in negative
controls. Each secondary reagent was confirmed to be species specific
by secondary antibody crossover (eg, mouse primary antibody followed by
anti-rabbit or anti–guinea pig secondary antibody).
Confocal Laser Scanning Microscopy and Image Analysis
Immunostained samples were examined by means of
confocal laser scanning microscopy using a Leica TCS SP equipped with
an argon/krypton laser with the appropriate filter spectra adjusted for
the detection of CY3 and CY5 fluorescence.
Single-connexin–labeled samples were used for semiquantification of
gap junctions. After the signal on top of the sample was observed,
images were collected using the x40 objective lens and zoom 1.0
computer setting so that each pixel represented 0.24
µm. Each recorded image consisted of 1024x1024 pixels, and
projection views of consecutive optical sections taken at 0.4-µm
intervals through the full thickness of endothelial
connexin signal were recorded for analysis. Mean thickness
of the vascular wall investigated was 6 µm. For double labeling,
images were taken using simultaneous dual-channel scanning.
Connexin labeling from the images was analyzed by using
procedures similar to those described and validated
previously.27 28 29
Images of single-connexin–labeled samples were analyzed by using QWIN image analysis software (Leica). For each animal, 1 injured plus 1 uninjured arterial ring was used. From each arterial ring, 4 randomly selected fields were analyzed. Software setting were kept constant in all animals. Mean values (±SDs) of (1) the area of individual immunolabeled gap junctions, (2) the number of immunolabeled gap junctions per 100 µm2 of luminal surface area, and (3) the total area of immunolabeled gap junction, expressed as the percentage of luminal surface area, were obtained for each experimental group.
The extent of connexin colocalization in the endothelia in each experimental group was also analyzed using double-connexin–labeled arterial rings. Projections of images were collected and split into 2 separate ("split") images corresponding to each connexin. Fifty immunolabeled spots were randomly selected from each double-label image, and the component connexins of each spot were determined by analyzing the split images. When a spot visible on the double-label image had a corresponding spot on each of the 2 split images (ie, both connexins were present), the sample was classified as showing colocalization. If a spot appeared on only 1 split image and not on the other, the sample was classified as containing 1 or the other individual connexin. For each experimental group, 2 images from each animal (ie, 100 gap junction spots) were analyzed. Results were expressed as the mean percentage (±SD) of gap junction spots showing fluorescence for each individual connexin that also showed fluorescence for the second connexin in each double-label combination.
Statistical Analysis
Data were compared statistically by means of ANOVA and Student’s
t test.
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Results |
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In animals injected with Evans blue dye after passage of PE50 tubing, the left carotid artery was homogeneously stained in blue, indicating damage to the endothelial layer. Blue staining ceased at the junction with the aortic arch. In contrast, the right carotid arteries (controls) were free of staining, and all 3 connexins were present in the endothelium. After injury, the pattern of expression of the 3 connexins was markedly altered; the sequence of changes observed for each connexin is illustrated in Figure 1
. Corresponding
data are summarized in Figure 2, and results of double labeling and connexin colocalization are shown in
Figures 3 and 4.
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Gap Junction Distribution and Connexin Expression in Normal
(Noninjured) Arterial Endothelium
En face views of the luminal surface after single labeling clearly
displayed the endothelial pavement, with punctate
connexin signal, typical of gap junctions, delineating the borders of
endothelial cells (Figures 1A
through 1C).
Overall, the 3 connexins shared a similar expression pattern, including
(1) the number of gap-junctional spots per unit area (Figure 2, top), (2) spot size (Figure 2, middle), and (3) percentage area
of connexin signal (Figure 2, bottom).
Double labeling enabled examination of the spatial relationship between
pairs of different connexins. Colocalization of 2 connexins within the
same spot was indicated by yellow fluorescence, which was due
to direct superimposition of red and green fluorescence. For
each of the 3 combinations, Cx37 with Cx40 (Figure 3A), Cx37
with Cx43 (Figure 4A), and Cx40 with Cx43 (Figure 4C),
>80% of the spots expressing 1 connexin also expressed the other (the
Table).
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Gap Junction Distribution and Connexin Expression After
Injury
Changes in gap junction distribution and connexin expression
observed after injury involved all 3 connexins (Figures 1 and 2). Immediately after injury, as the endothelium
was denuded, fluorescence signal at the luminal surface
attributable to endothelial gap junctions was absent.
Endothelial marking and electron microscopy
demonstrated reappearance of endothelial cells from 7
days onward (Figures 5 and 6). Sequential confocal observation
revealed that each of the 3 connexins had distinct spatial and temporal
expression patterns (Figures 1D through 1L). For each connexin,
the change was statistically significant between different time points
(P<0.01).
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Single-Connexin Labeling
For Cx37, immunolabeled spots were sparsely scattered throughout
the regenerating endothelium 7 days after injury
(Figure 1D) compared with the abundant expression in the intact
artery (Figure 1A). In addition, spot size and total
gap-junctional area were smaller (day 7 versus control,
P<0.01; Figure 2). This pattern abruptly changed at
the junctional zone with the aortic arch, where clusters of
endothelial cells abundantly labeled with Cx37, as seen
in the uninjured artery, were found. Fourteen days after injury, Cx37
spots were unevenly distributed (Figure 1G), and spot number,
size, and area were increased significantly (P<0.01). By
day 28 (Figure 1J), Cx37 spot number had returned to and Cx37
spot size and area exceeded levels of the uninjured artery (day 28
versus control, P<0.01 for Cx37 spot size and
P<0.05 for Cx37 area). Cx40 followed a similar expression
pattern after injury. Figure 1E, taken from the junctional zone
of the artery with the aorta 7 days after injury, shows clusters of
Cx40-rich cells from the uninjured area with only few spots in the
bordering injured zone. During recovery, the number of Cx40 spots and
total gap-junctional area in the injured artery progressively increased
(Figures 1H, 1K, and 2, P<0.01). However,
although the average size of Cx40 spots increased initially (days 7
versus 14, P<0.01), unlike Cx37, no additional increase
occurred after 14 days.
The temporal pattern of Cx43 expression differed from that of the other
connexins but in a cell type–related manner. Seven days after injury,
spindle-shaped cells expressing Cx43 were unevenly distributed on the
luminal side of the injured artery, below the level of the
endothelium (Figure 1F). In addition to the
punctate label along the cell border, Cx43 signal was apparent in the
cytoplasm, especially concentrated at the perinuclear region. Judging
from their morphological characteristics, location, and relationship
with the endothelial cells, as demonstrated by double
labeling for Cx43 and von Willebrand factor (Figure 5),
these spindle-shaped cells were identified as
subendothelial smooth muscle cells. Only sparse Cx43
spots were seen outside these spindle-shaped cells, which abruptly
disappeared after day 7 (Figures 1I and 1L). Because Cx43 signal
from cellular processes of smooth muscle cells was difficult to
distinguish from those of endothelial cells, no
analysis was performed at this time point. After day 7,
expression of Cx43 in the regenerating endothelium
increased in a temporal pattern similar to that of Cx37 (Figures 1I, 1L, and 2).
Double-Connexin Labeling
Double labeling of the injured artery at all stages demonstrated
that the presence of >1 connexin in the gap-junctional spots was a
common feature in regenerating endothelium. Figures 3B through 3D contain typical images from injured artery double
labeled for Cx37 and Cx40. Seven days after injury, >60% of the
sparsely observed spots expressed both connexins. In general, spots
expressing 1 connexin and not the other were smaller than those
expressing both. In the connexin-rich endothelial cells
at the junction between the injured carotid artery and uninjured aortic
arch, extensive colocalization was apparent (Figure 3B). As the
regenerating endothelium progressively developed gap
junctions 14 and 28 days after injury (Figures 3C and 3D), the
extent of colocalization increased to >80% (the Table).
A similar extent of colocalization was found with combinations of Cx43
plus Cx37 and Cx43 plus Cx40 (Figure 4). Seven days after
injury, the junctional zone between the injured carotid artery and
aortic arch contained abundant Cx43-expressing smooth muscle cells on
the carotid side (Figure 4B). As in the corresponding
single-labeling experiments, endothelial Cx37 and Cx40
labeling was sparse; however, the majority of Cx37 and Cx40 spots also
expressed Cx43. As the Cx43-expressing smooth muscle cells became
concealed by the regenerating endothelium, the images
of Cx37 with Cx43 and of Cx40 with Cx43 developed in a pattern similar
to that of Cx37 with Cx40 (Figure 4D).
Electron Microscopic Examination
In the intact carotid artery endothelium, gap
junctions were frequently seen at cell borders (Figure 6A).
Immediately after injury, the endothelium was so
severely damaged that no intact cells remained on the internal elastic
lamina (Figure 6B). From this stage, the
endothelium gradually recovered. Seven days after
injury, endothelial cells covered substantial areas
(but not all) of the previously damaged luminal surface. At this stage,
junctional borders between adjacent endothelial cells
were limited in size and had occasional tiny gap junctions (Figures 6C and 6D). These findings thus excluded the possibility that at
7 days after injury the reduction of endothelial
connexin expression, as observed in the immunolabeling experiments, was
due to the absence of endothelial cells. Fourteen days
after injury, gap junctions were more numerous and larger (Figure 6E). This trend continued, and by day 28 large, abundant gap
junctions were observed (Figure 6F).
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Discussion |
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The present study demonstrated that after regeneration of injured endothelium, gap junction number initially declines but subsequently recovers to the level found in the uninjured artery, that this process involves all 3 connexins (Cx37, Cx40, and Cx43), and that colocalization of different connexins to the same gap junction is a common feature beginning early in the regeneration process. Within this framework, there are distinct differences in the changes in relative expression levels of the 3 connexins. Whereas the amount of Cx40 signal recovers to the same level, the amount of Cx37 and Cx43 signal reaches higher levels than in the uninjured artery as a result of the formation of larger gap-junctional spots containing these connexins. These findings, demonstrated by en face immunoconfocal viewing of the endothelium and complementary electron microscopy, substantially expand current knowledge of gap junction biology in the healing vascular wall.
Note that immunoconfocal microscopy, as applied here, does not provide
quantitative data on the absolute amounts of connexin expressed in the
endothelial cells but, as established by previous
studies,27 28 29 does give a reliable guide to the relative
level of a given connexin assembled in the form of gap junctions. The
data are thus distinct from those obtained by quantification of Western
blots, in which the relative overall amounts of total (cytoplasmic and
gap-junctional) connexin over the tissue as a whole are measured. Our
finding that gap junctions were tiny and rare initially after injury
and progressively grew in size and number during regeneration is
consistent with findings of previous studies that used
freeze-fracture electron microscopy on mechanically injured rabbit
carotid artery and rat aorta.26 30 However, these earlier
experiments gave no information about the nature of the connexins
present. We showed that all 3 connexins are affected and respond in
distinct ways. Connexin expression in mechanically wounded
endothelial cells in vitro is reported to differ
between microvascular (bovine adrenal cortex) and large-vessel
endothelial cells (bovine aorta and pulmonary
artery); increased Cx43 has been reported in the
former19 31 but not the latter.18 19 If the
in vitro models faithfully reflect in vivo denudation, then the changes
in the carotid artery investigated in this study would be expected to
conform to those of large-vessel cells. However, our results showed
that Cx43 expression is elevated after 4 weeks in vivo. Apart from
mechanical injury, other factors are reported to affect
endothelial connexins. For example, increased
mechanical load enhances Cx43 expression,23 and treatment
with tumor necrosis factor-
, a cytokine promoting
endothelial cell migration, leads to upregulation of
Cx43 and downregulation of Cx37 and Cx40 in human umbilical vein
endothelial cells.25 In addition, Cx43 and
Cx37 in cultured bovine aortic endothelial cells are
reported to be differentially regulated by cell density and growth
status21 ; although levels of Cx43 mRNA and protein were
high in subconfluent cells and low in confluent cells, the opposite
applied to Cx37 mRNA, which was present only in low amounts until
the cultures became confluent. In contrast, in the present study,
Cx37 and Cx43 spots increased progressively during the course of
endothelial regeneration in vivo. These comparisons
emphasize that expression patterns of connexins in culture systems may
not mimic those occurring in vivo and that caution is required in
extrapolation of data from in vitro studies to whole vascular
tissues.
Apart from in vitro studies, investigation of intact vascular tissues demonstrates that connexin expression is influenced by a variety of factors. For example, the precise levels of expression of the 3 endothelial connexins depends in part on vascular site,11 12 13 14 and the similar signal levels for the 3 connexins in uninjured rat carotid artery endothelium, distinct from the patterns previously reported in aorta, pulmonary artery, and coronary artery of the same animal, are in keeping with this finding. In a recent study examining rat aorta and its branching arteries, high levels of endothelial Cx43 gap junctions were associated with areas facing turbulent flow.32 Our finding that expression of endothelial Cx43 is enhanced 4 weeks after injury is consistent with this result, because the luminal profile of an injured artery favors generation of turbulent flow.
An important finding of the present study is that in regenerating endothelium, gap junctions are commonly composed of >1 connexin, even from the earliest stage when the junctions are small and sparse. Colocalization of connexins has been reported in endothelial cells of microvessels and large vessels.11 33 In addition, our previous work in rat aorta and pulmonary artery demonstrated that individual endothelial gap-junctional plaques contain up to 3 connexins.13 14 The present study showed that over the regrowth of the monolayer, different connexins tend to mix within individual gap junctions. Furthermore, the relative amounts of individual connexins in the gap junctions during this process is time dependent, as indicated by the finding that colocalization occurs more frequently during the later stages. Although the connexin makeup of individual channels cannot be determined by confocal microscopy, the results of semiquantification and colocalization analysis suggest that the distribution of each connexin in individual gap junctions containing >1 connexin is not uniform. Given that most gap junctions contain >1 connexin, if the connexins were homogeneously distributed, then the dimensions of gap junctions obtained from immunolabeling using antibodies against the component connexins at different stages after injury would be expected to show a similar trend. In contrast, we found that although Cx37 and Cx43 spots continued to increase in size, Cx40 spots remained constant after 14 days.
Overexpression of connexins after injury raises the possibility that gap-junctional intercellular communication properties of the injured artery differ from those of the uninjured artery. In vitro studies have established that each connexin endows gap-junctional channels with unique properties.8 15 34 35 36 Although the precise role of each connexin in vivo remains unclear, reports from connexin gene knockout studies give some useful clues. Deletion of Cx43 in the mouse leads to postnatal death because of a cardiac anomaly, deletion of Cx37 leads to female sterility, and deletion of Cx40 leads to abnormal cardiac conduction, but in all cases no obvious morphological defect is observed in the vascular endothelium.37 38 39 40 However, when Cx37 and Cx40 are both deleted from the genome, the animals die perinatally and defective vascular integrity is prominent.41 Thus, although there seems to be some capacity for functional compensation by remaining connexins when 1 member is absent, lack of 2 connexins compromises endothelial function. However, the possibility that lack of a single endothelial connexin predisposes to long-term vascular disease cannot be excluded.
In conclusion, rat carotid arterial endothelium retains the ability to generate the 3 connexin proteins into gap junctions after mechanical injury. The distinct patterns of expression, distribution, and organization of these connexins into gap junctions suggest a capacity to modulate communication properties between neighboring cells during different phases of the healing process. The different responses of the individual connexins indicate that each connexin in the endothelial cell is subjected to specific regulation during this process.
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Acknowledgments |
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This work was supported by grants NSC-89-2314-B-195-006 from the National Science Council of Taiwan and MMH-8702 from the Medical Research Department of Mackay Memorial Hospital. We thank Drs E. Dupont and S. R. Coppen for assistance in the purification of anti-connexin antisera. N.J.S. acknowledges support from the European Commission.
Received October 8, 1999; accepted December 21, 1999.
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