ARTICLE |
Correspondence to: Cheng-Ho Tsai, Internal Medicine, Mackay Memorial Hospital, 92, Sec 2, North Chung San Road Taipei 10449, Taiwan. E-mail: cht7678@ms2.mmh.org.tw
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Summary |
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We investigated endothelial gap junctions and their three component connexins, connexin37 (Cx37), Cx40, and Cx43, during growth and senescence in rat aorta by en face immunoconfocal microscopy and electron microscopy. Gap junction spots labeled by specific antisera against Cx37, Cx40, and Cx43 were quantified at 1 day, 7 days, 28 days, 16 months, and 20 months of age, and the relationship between the connexins was examined by co-localization analysis. At birth, all three connexins were abundantly expressed; the number and total area of connexin spots then declined within 1 week (p<0.05 for each connexin). From 1 week, each connexin showed a distinct temporal expression pattern. Whereas Cx43 signal decreased progressively, Cx37 signal fluctuated in a downward trend. By contrast, Cx40 maintained an abundant level until
20 months of age (
20 months vs 28 days, p<0.05 for number and total connexin signal area). These patterns were associated with changes in endothelial cell morphology. Double-label analysis showed that the extent of co-localization of connexins to the same gap junctional spot was age-dependent [>70% at birth and 28 days old; <70% at later stages (p<0.05)]. We conclude that expression of the three connexins in aortic endothelium is age-related, implying specific intercellular communication requirements during different stages after birth.
(J Histochem Cytochem 48:13771389, 2000)
Key Words: gap junction, connexin, endothelial cells, age
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Introduction |
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Vascular endothelial cells originate from mesodermal angioblasts. During early embryonic development these isolated cells assemble into cords, which then differentiate into recognizable endothelial cells interconnecting with one another to form a thin single layer surrounding a central lumen (
Gap junctions are cell membrane protein channels clustered at cellcell junctions. Each channel is composed of two hemichannels (named connexons), and each hemichannel comprises six connexin subunits linked side by side to leave a central canal. The two connexons align end to end across the intercellular space, with the connecting canals bridging the cytoplasmic compartments of adjacent cells, allowing exchange of ions and small molecules (<1,000 daltons), and thus direct intercellular communication (
Current knowledge about endothelial gap junctions is growing rapidly, these communication conduits having been implicated in a variety of endothelial activities, such as maintenance of monolayer topology, coordination of vasomotor responses, regulation of angiogenesis, and endothelial growth and senescence (
In view of the important roles of gap junctions in endothelial cell function, knowledge of the endothelial gap junctions and connexins in different stages of life is essential to interpretation of experimental data and for understanding vascular biology and the related pathologies. This study therefore set out to investigate the spatial and temporal expression pattern of endo-thelial gap junctions and connexins in rat aorta after birth.
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Materials and Methods |
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Samples and Tissue Processing
Samples of aorta from SpragueDawley rats at the ages of 1 day, 7 days, 28 days, 16 months, and 20 months after birth were investigated in this study (total 35 rats; seven animals in each age group). Male rats were used for those at age of 28 days or older. These animals were anesthetized with sodium pentobarbital (50 mg/kg IP) and perfused retrogradely, via a catheter in the abdominal aorta, with heparinized PBS (10 U/ml) followed by phosphate-buffered 2% paraformaldehyde (pH 7.4) for 10 min. For groups below 28 days of age, after ether inhalation, the animals received perfusion-fixation via direct intracardiac injection. In all animals the descending thoracic aortae were dissected and cut into transverse rings for rapid freezing in isopentane at -160C. The samples were then stored under liquid nitrogen before immunolabeling. For thin-section electron microscopy, selected arterial samples were prepared by standard procedures. The work was conducted in accordance with the ROC Animal Protection Law (Scientific Application of Animals), 1998.
Generation, Affinity-purification, and Characterization of Anti-Cx37 Antibody
A polyclonal Cx37 antibody was generated, followed by affinity-purification and characterization using the method previously described (
The specificity and crossreactivity to other connexins of the purified anti-peptide antibody were tested by Western blotting and immunolabeling using HeLa cells, kindly donated by Professor Klaus Willecke [University of Bonn, Germany (
Immunofluorescence Labeling of Endothelial Connexins
Anti-connexin Antibodies.
In addition to the Cx37 antibody, two other antibodies were used for immunofluorescence detection of Cx40 and Cx43. The Cx40 polyclonal antiserum was produced in guinea pigs against a synthetic peptide corresponding to residues 256270 of the cytoplasmic C-terminal tail of rat Cx40 [designated V15K(GP319)] and was affinity-purified (
Secondary Antibody/Detection Systems. Donkey anti-rabbit, anti-guinea pig, and anti-mouse immunoglobulin conjugated either 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 two connexins, one CY3-conjugated antibody and one CY5-conjugated antibody were used in combination.
Immunolabeling of Connexins.
For single labeling of one connexin type, the unfixed HeLa cells were first immersed in -20C methanol for 5 min. All cells and the perfusion-fixed aortic rings were rinsed in PBS for 5 min, blocked in 0.5% BSA (15 min), and incubated with anti-Cx37 (1:200), anti-Cx40 (1:100), or anti-Cx43 (1:500) at 37C for 2 hr. The samples were then treated with CY3-conjugated secondary antibody (1:500, room temperature, 1 hr). In experiments in which two of the three connexins were simultaneously localized in the same samples, incubation was with a mixture of anti-Cx37 plus anti-Cx40, anti-Cx37 plus anti-Cx43, or anti-Cx40 plus anti-Cx43, followed by incubation with a mixture of the two corresponding species-specific secondary antibodies (CY3 and CY5; 1:500). Finally, the arterial rings were cut open and mounted for en face viewing of endothelial cells. All experiments included rat heart sections as positive controls (
Confocal Laser Scanning Microscopy and Image Analysis
Immunostained samples were examined by 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 and the luminal surface area of individual cells. After the signal on the top of the sample was observed, the images were collected using the x40 objective lens and zoom 1.0 computer setting so that each pixel represented 0.24 µm. Each image recorded consisted of 1024 x 1024 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. The mean thickness of the vascular wall investigated was 6 µm. For double labeling, the images were taken using simultaneous dual-channel scanning. Analysis of connexin labeling from the images was conducted following similar procedures to those described and validated previously (
Analysis of images from single-connexin-labeled samples was undertaken using QWIN image analysis software (Leica; Heidelberg, Germany). For each animal, two arterial rings were used. From each arterial ring, two randomly selected fields were analyzed. The value of the software setting was kept constant in all animals. Mean values (±SD) of the following were obtained for each age group: (a) the area of individual immunolabeled gap junctions; (b) the number of immunolabeled gap junctions per 100-µm2 luminal surface area; (c) the total area of immunolabeled gap junctions, expressed as percentage of the luminal surface area; and (d) the luminal surface area per cell. For luminal surface area, 20 endothelial cells were randomly selected from each image.
The extent of connexin co-localization in the endothelia in each age group was also analyzed using double-connexin-labeled arterial rings. Projections of images were collected and split into the two separate ("split") images corresponding to each of the connexin types. 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 two split images (i.e., both connexins were present), it was classified as showing co-localization. If a spot appeared on just one of the split images and not on the other, it was classified as containing one or other of the individual connexin types. For each age group, two images from each animal (i.e., 100 gap junction spots) were analyzed. Results were expressed as mean percentages (±SD) of gap junction spots showing fluorescence for each individual type of connexin that also showed fluorescence for the second connexin type, in each double-label combination.
Data were compared statistically by ANOVA and t-test.
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Results |
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Characterization of Anti-Cx37 Antibody
Western blotting analysis showed that the anti-Cx37 antibody recognized a single band at 37 kD in HeLa-37 cell membrane preparations (Fig 1). This band, absent in the corresponding preparations of HeLa-W, HeLa-40, HeLa-43, and HeLa-45 cells, was markedly inhibited by preincubation of the antibody with the peptide to which it was raised.
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Immunoconfocal microscopy of HeLa-37 cells incubated with the anti-Cx37 antibody demonstrated distinct punctate labeling at cellcell borders, which was abolished by peptide inhibition (Fig 2). No positive signal was apparent when the antibody was tested on HeLa-W, HeLa-40, HeLa-43, and HeLa-45 cells (Fig 2).
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Localization of Endothelial Connexins by Confocal Microscopy: Single-labeling Experiment
En face views of the luminal surface after single labeling with each of the three anti-connexin antibodies clearly displayed the endothelial cells outlined with punctate connexin signal, typical of gap junctions, at the cell borders. Representative images from each age group are shown in Fig 3.
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Quantitative analysis of the immunoconfocal images showed that for each connexin type the expression patterns varied between different age groups, as determined by (a) the number of gap junctional spots per unit area, (b) the average spot size, and (c) the total gap junction area, expressed as percentage area of connexin signal (p<0.01 for all the three values of each connexin). The data are summarized in Fig 4.
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At birth, each of the three connexinsCx43, Cx37, and Cx40shared a similar expression pattern. They were abundant and were more or less homogeneously distributed over the endothelium (Fig 3A3C), reflecting the small cell size at this stage (see below). However, within 1 week the expression patterns changed (Fig 3D3F). The three connexins were less evenly distributed and the gap junction spot number and total area declined (1 day vs 7 days, p<0.01 for each connexin; Fig 4). With respect to the average spot size during this period, that of Cx43 decreased but those of Cx37 and Cx40 remained similar (1 day vs 7 days, p<0.05 for Cx43; p>0.05 for either Cx37 or Cx40). After the first week, each connexin had a distinct expression pattern (Fig 3G3O). For Cx43, the amount of labeling decreased progressively (Fig 3G, Fig 3J, and Fig 3M), as reflected by the declining numbers of spots and total gap junction areas (7 days vs 16 months or older, p<0.05), as well as a similar trend of reduction in the average spot sizes (Fig 4). Unlike Cx43, Cx37 decreased in a zigzag manner after 7 days of age (Fig 3H, Fig 3K, and Fig 3N). The number of spots and the total gap junction area of Cx37 had an initial decline at 28 days (7 days vs 28 days, p<0.01), followed by an increase at 16 months (28 days vs 16 months, p<0.05) and a final fall afterwards (16 months vs 20 months or older, p<0.01). However, the average spot size of Cx37 increased (28 days vs 16 months or older, p<0.05; Fig 4). In addition to the level of expression, one common finding for both Cx43 and Cx37 was that they were unevenly distributed after 1 week of age. By contrast, at 28 days of age, Cx40 resumed the homogeneous distribution seen at birth, and this pattern persisted until at 20 months of age or older, when the labels became heterogeneously distributed (Fig 3I, Fig 3L, and Fig 3O). Considering the level of expression, Cx40 was abundant at 28 days and 16 months of age. The values of total area of Cx40 labels of the two age groups were comparable, although the number of spots was higher in the former and the average spot size was larger in the latter (Fig 4). After 16 months of age, the expression of Cx40 was downregulated (16 months vs 20 months or older, p<0.05 for the number, average spot size, and total area of Cx40 gap junctions).
It should be mentioned that variation of connexin expression existed among animals of the same age group. In the oldest group, in particular, the number of spots and total area of Cx37 and Cx40 showed a wide variation. Although in general the values of each connexin in the group of 20 months of age were reduced compared to the corresponding 16-month-old animals, some animals of the former group showed more apparent downregulation than did others, irrespective of age. On the other hand, a deep downregulation of Cx37 or Cx40 in animals of the oldest group was not necessarily associated with the same degree of change of the other connexin.
Morphological Changes of the Aorta and Endothelial Cells
The dimensions of the thoracic aorta varied considerably among different age groups. For example, at birth the caliber of the middle portion of the descending thoracic aorta was 1.1 ± 0.1 mm. It rapidly increased to 1.3 ± 0.2 mm at 7 days of age and 2.6 ± 0.4 mm at 28 days. After this, the size increased slowly so that at the age of 16 months and 20 months it measured 3.0 ± 0.5 mm and 3.1 ± 0.4 mm, respectively. Accompanying the increase of aortic lumen was the growth of endothelial cell size. Because immunostained connexin spots were located at cellcell junctions, the borders of individual cells can be traced. This enabled calculation of the luminal surface area of individual cells. For this purpose, images single-labeled for Cx40 were used because, as mentioned above, Cx40 was abundantly expressed through the time series until the oldest stage. Measurement from endothelium rich in Cx40 labels at the middle portion of the descending thoracic aorta showed that the size of individual endothelial cells followed a similar increase as the macroscopic change of aorta. The luminal surface area per cell was 143.2 ± 48.2 µm2 at birth, 158.7 ± 28.7 µm2 at 7 days, 336.8 ± 91.5 µm2 at 28 days, 358.2 ± 92.4 µm2 at 16 months, and 364.2 ± 87.6 µm2 at 20 months or older (p<0.01 among different time points).
Double-labeling Experiment
Double labeling enabled examination of the spatial relationship between pairs of different connexins. Co-localization of two connexins within the same spot was detected as yellow fluorescence due to direct superimposition of red and green colors. For each of the three combinationsCx37 with Cx40, Cx37 with Cx43, and Cx40 with Cx43although variations existed between different age groups, at least 50% of the spots positive for one connexin type were also positive for the other (Table 1). In general, co-localization was >70% at birth, declined to <70% at 1 week of age, and recovered to >70% at 28 days of age, followed by a stepwise fall afterward (p<0.05 for each pairing connexins among different time points). Typical images of the three combinations for each age group are shown in Fig 5.
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Electron Microscopic Examination
At birth, the endothelium was separated from the underlying medial smooth muscle by a thin belt-like structure, which had a similar appearance to those dividing the medial smooth muscle into layers and developed later into the internal elastic lamina (Fig 6A). In general, the outline of endothelial cells was smooth on the luminal side but irregular on the lateral side, where rather convoluted junctional borders formed between neighboring cells. Although most of the junctional borders were typically made by intimate apposition of adjacent cell membranes (Fig 6B), interdigitation with intermittent contact between cell processes of bordering cells, which may indicate loosening of the junction for migration and/or proliferation of the cells, was not infrequently seen (Fig 6C). At this stage, cell nuclei were frequently gathered, reflecting the small size of the cells and high cellularity of the endothelium (Fig 6D). Such a configuration of the endothelium did not change much at 7 days of age unless the cell increased in dimension. Gap junctions of various sizes were frequently seen at these two stages. Usually small gap junctions were seen at the convoluted junctional borders, and large ones were located at less convoluted borders (Fig 6E and Fig 6F).
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By contrast, at 28 days, the separation between endothelial cells and the underlying medial smooth muscle increased, owing to the growth of the internal elastic lamina (Fig 6G). At the luminal interface, although the cells still had a smooth outline, the cell nucleus was not frequently seen because of lateral expansion of the cell body, which became flattened. At the lateral side, in addition to the convoluted borders, long curvilinear borders containing tandem gap junctions of various sizes were common (Fig 6G).
The morphology of the endothelium changed markedly by the age of 16 months (Fig 7). A substantial portion of the cells had an irregular luminal outline, and abrupt protrusion of the cell body, which contained clusters of electron-dense inclusions in the cytoplasm, towards the lumen was not uncommon (Fig 7A and Fig 7B). In addition, rather than lying on the subepithelium, some of the cells discharged processes into the subendothelium like roots of a tree (Fig 7B). In parallel, long, curvilinear cellcell junctional borders were less common. However, gap junctions were still abundant (Fig 7A). Rarely, annular gap junctions were found inside the cell (Fig 7C). From this age onward, the appearance of the endothelium was maintained, although gap junctions became fewer and heterogeneously distributed.
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Discussion |
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This study shows that rat aortic endothelium at different stages of life has distinct patterns of gap junction distribution and connexin expression involving all three connexins (Cx37, Cx40, and Cx43), and that co-localization of these different connexins to the same gap junction is a common feature after birth. Within this framework, there are age-related differences in the relative expression levels of the individual connexins and their assembly into gap junctions. In general, all three connexins are maximally expressed at birth, followed by a decline within 1 week, and they are downregulated in old age. Between these two extremes, individual connexins behave differently. For example, unlike the progressive decline found with Cx43, Cx40 maintains a high level of expression until the oldest stage examined, while Cx37 fluctuates throughout the course. These findings, demonstrated by en face immunoconfocal viewing of the endothelium and complementary electron microscopy, substantially expand current knowledge of endothelial gap junctions in postnatal development, growth, and senescence.
Although endothelial cells are known to express up to three members of the connexin family, investigation of gap junction distribution and connexin expression in this cell type by traditional immunofluorescence microscopy using sectional views faces a variety of pitfalls. These mainly stem from the monolayer topology of endothelial cells. For example, the extent of the endothelium is not easily discriminated from the remaining vascular wall, and only limited sample size per section can be obtained for analysis (
Interpretation of the dynamic expression of endothelial gap junctions and connexins after birth should consider the characters of endothelial cells at each stage of life. One distinct change of the cells related to age observed in the present study is the cell morphology, including the size, as clearly seen from both the ultrastructural examination and the image analysis. The latter shows that the luminal cell surface area grows by more than twofold during the first month after birth and then increases slowly thereafter. Accordingly, interpretation of the age-related change of the connexins can be divided into two parts, i.e., the changes during and after the first month.
Our result showing that the three connexins are differentially regulated as the cells grow rapidly is consistent with previous reports investigating gap junction distribution and connexin expression during postnatal development in other cell types or organs, such as vascular smooth muscle (
On the other hand, our findings indicate that, during postnatal development, the expression patterns of individual endothelial connexins in situ are not the same as observed in cell culture, in which Cx37 and Cx43 expression is inversely related to cell density (
An important finding from the present study is that all the three connexins are downregulated at the later stage of life. In vitro study has shown that expression of Cx43 declines in senescent endothelial cells (
In conclusion, rat aortic endothelial cells form various numbers of gap junctions with different content of Cx37, Cx40, and Cx43 according to age. The differential expression of the three connexins indicates that intercellular communication in the endothelium is not stationary after birth and suggests that complicated mechanisms and interactions are involved for regulation of individual connexins. Downregulation of the three connexins at old age implies a possible deficiency of intercellular communication in the senescent cells, with a potential role in endothelial dysfunction.
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Acknowledgments |
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Supported by grants NSC-89-2314-B-195-006 from the National Science Council, Taiwan and MMH-8702 from the Medical Research Department of the Mackay Memorial Hospital, Taiwan. NJS acknowledges support from the European Commission.
We thank Dr Emmanuel Dupont and Dr Steven R. Coppen (NHLI; Imperial College, London, UK) for assistance in the purification of the anti-connexin40 antiserum, Ray-Ching Hong and Shiu-Ching Chen for assistance with electron microscopy, and Jain-Ming Cho for assistance with the animal work.
Received for publication February 15, 2000; accepted April 26, 2000.
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