Journal of Histochemistry and Cytochemistry, Vol. 48, 1377-1390, October 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Age-related Alteration of Gap Junction Distribution and Connexin Expression in Rat Aortic Endothelium

Hung-I Yeha, Hao-Min Changa, Wen-Wei Lua, Yi-Nan Leea, Yu-Shien Kob, Nicholas J. Seversc, and Cheng-Ho Tsaia
a Departments of Internal Medicine and Medical Research, Mackay Memorial Hospital, Taipei Medical College, Taipei, Taiwan
b National Heart and Lung Institute, Imperial College School of Medicine, London, United Kingdom and The First Cardiovascular Division, Department of Internal Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan
c National Heart and Lung Institute, Imperial College School of Medicine, London, United Kingdom

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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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 48:1377–1389, 2000)

Key Words: gap junction, connexin, endothelial cells, age


  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 (Cleaver and Krieg 1999 ). From this stage on throughout postnatal growth, maturation, and senescence, the endothelium mediates the interaction between the blood and the vascular wall, participating in the regulation of a variety of critical functions, e.g., vascular tone and hemostasis (Stary et al. 1992 ). In each of these processes, integrity of the function of this monolayer under specific physiological conditions is essential for maintenance of homeostasis. Such a versatile role of endothelium requires coordination of the activity between individual cells, in which several signaling mechanisms are involved, including gap junctional intercellular communication.

Gap junctions are cell membrane protein channels clustered at cell–cell 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 (Severs 1995 ; Bruzzone et al. 1996 ; Kumar and Gilula 1996 ). The properties of gap junctional channels are determined by the component connexins, which belong to a multigene family. Of the 16 members thus far identified in mammalian cells, Cx37, Cx40, and Cx43 are known to be variously expressed in different types of endothelial cell (Van Rijen et al. 1997 ; Yeh et al. 1997a , Yeh et al. 1998 ; Hong and Hill 1998 ; Ko et al. 1999 ). For example, in adult rat aorta, previous work has shown that these three connexins are expressed in the endothelium, commonly assembled into the same gap junction plaque (Yeh et al. 1997a , Yeh et al. 1998 ). By contrast, in intramural coronary arteries of the same animal, whereas Cx37 or Cx40 has a similar expression to that of the aorta, Cx43 is barely detectable (Yeh et al. 1997a , Yeh et al. 1998 ; Hong and Hill 1998 ).

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 (Larson 1988 ; Pepper et al. 1992 ; Xie and Hu 1994 ; Christ et al. 1996 ; Larson et al. 1997 ). In addition, the specific pattern of connexin expression is differentially affected by physical and chemical factors, e.g., mechanical load and injury, blood sugar level, growth factors and cytokines (Pepper and Meda 1992 ; Xie and Hu 1994 ; Cowan et al. 1998 ; Kuroki et al. 1998 ; Van Rijen et al. 1998 ). However, information on endothelial connexins in different stages of postembryonic life is fragmentary. Although in vitro studies have shown that expression of endothelial connexins is not stationary during growth and senescence, these reports are limited to examination of one or two connexins (Xie and Hu 1994 ; Larson et al. 1997 ). In addition, results from in vitro studies seldom faithfully reflect those occurring in vivo, as revealed, for example, in our recent report examining connexins during endothelial regeneration after denudation injury (Yeh et al. in press ).

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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Samples and Tissue Processing
Samples of aorta from Sprague–Dawley 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 (Yeh et al. 1998 ). In brief, the antibody, designated Y16Y(R382), was raised in a rabbit, using the commercial service of Research Genetics (Huntsville, AL), against the peptide corresponding to residues 266–281 of the cytoplasmic C-terminal tail of rat Cx37. The immune serum was affinity-purified against the peptide coupled to epoxy-activated Sepharose 6B (Pharmacia Biotech; Herts, UK). The antibody was eluted from the column using 0.1 M glycine, pH 2.4.

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 (Elfgang et al. 1995 )], which were genetically manipulated to express mouse Cx37 (HeLa-37), Cx40 (HeLa-40), Cx43 (HeLa-43), or Cx45 (HeLa-45). The wild-type cells (HeLa-W) served as controls. For Western blotting, alkaline membrane preparations of cultured cells were prepared. For immunolabeling, the cells were grown on glass coverslips. Procedures of Western blotting and cell culture followed those described previously (Yeh et al. 1998 ).

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 256–270 of the cytoplasmic C-terminal tail of rat Cx40 [designated V15K(GP319)] and was affinity-purified (Yeh et al. 1998 ). For Cx43, a mouse monoclonal antibody (MAb) was purchased from Chemicon (Temecula, CA). These two antibodies have previously been confirmed, using HeLa cell transfectants as described above, to be isotype-specific and not to crossreact with other endothelial connexins (Yeh et al. 1998 ).

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 (Yeh et al. 1997a ) and omission of primary antibody as negative controls. Each secondary reagent was confirmed to be species-specific by secondary antibody cross-over (e.g., mouse primary antibody followed by anti-rabbit and/or anti-guinea pig secondary).

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 (Green et al. 1993 ; Blackburn et al. 1995 ; Yeh et al. 1997b ; Yeh et al. in press ).

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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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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Figure 1. Characterization of Cx37 anti-peptide antibody by Western blotting. The positions of molecular mass standards are indicated at left. The antibody binds specifically to a 37-kD band in HeLa-37 transfectants. The binding was inhibited by addition of the peptide. No such bands are detected in HeLa-40, HeLa-43, HeLa-45, or HeLa wild-type controls. Note that other nonspecific bands of higher molecular weights are consistently present in all the HeLa cells, reflecting nonspecific binding to irrelevant proteins.

Immunoconfocal microscopy of HeLa-37 cells incubated with the anti-Cx37 antibody demonstrated distinct punctate labeling at cell–cell 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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Figure 2. Characterization of Cx37 anti-peptide antibody by immunocytochemistry. The antibody gives prominent labeling, which is located both at cell–cell borders, typical of gap junctions, and around the nuclei, only in HeLa-37 transfectants (A). The specificity of this antibody is confirmed by absence of labeling in HeLa wild-type, HeLa-40, HeLa-43, and HeLa-45 cells (B–E). The positive labeling in HeLa-37 cells is abolished by peptide inhibition (F). Note that in A and F the signal of Cx37 is superimposed on the phase-contrast images to facilitate cellular localization of the labeling. Bar = 20 µm.

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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Figure 3. (A–D) Confocal images illustrating the chronological changes in pattern of expression of Cx43, Cx37, and Cx40 in rat aortic endothelium during different stages of postnatal life, as revealed by en face viewing after single labeling. Typical images for each connexin at the time points examined are displayed except for images N and O, which come from animals with lower expression level of Cx37 or Cx40, compared to the other animals of the same age group. Note that when the connexin spots are abundant, the entire border of endothelial cells can be easily defined by linking the spots around the individual cells. All images, oriented in parallel to the long axis of the artery with the cephalic side up, are at the same magnification. d, day; m, month. Bar = 30 µm.

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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Figure 4. Immunoconfocal analysis of gap junction spots detected by antibodies against Cx43, Cx37, and Cx40. Upper histogram, number of gap junction spots per unit area of the luminal surface. Middle histogram, mean size of gap junction spots. Lower histogram, total gap junctional area per unit area of luminal surface. Although each connexin has a distinct temporal pattern of change, the number and total area of gap junction spots, as detected with each antibody, are highest at birth and lowest at the oldest age examined. All comparisons are made between one time point and the following one. In upper and lower histogram, unless marked, p<0.01 for each comparison. Stars, p>0.1; +, p<0.05.

At birth, each of the three connexins—Cx43, Cx37, and Cx40—shared a similar expression pattern. They were abundant and were more or less homogeneously distributed over the endothelium (Fig 3A–3C), reflecting the small cell size at this stage (see below). However, within 1 week the expression patterns changed (Fig 3D–3F). 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 3G–3O). 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 cell–cell 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 combinations—Cx37 with Cx40, Cx37 with Cx43, and Cx40 with Cx43—although 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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Figure 5. En face confocal view of the endothelium double labeled for two connexins. The signals of individual connexin are in red or green. Each color is denoted and the corresponding age marked at the upper left of each image. At birth (A), both Cx43 (red) and Cx40 (green) are abundant, and most of the spots look yellowish due to superimposition of red and green colors of different intensity. However, spots of pure red or green color still can be seen (arrows). Image B is obtained from an animal of 7 days old. Both Cx37 (red) and Cx43 (green) spots are not as regularly distributed as seen in A, although most of the spots still contain red and green elements. At 28 days (C) and 16 months (D) of age, co-localization of two connexins is commonly seen along the border of cells, the size of which is apparently different from those in A and B. Images E and F come from different animals of 20 months or older of age. In E, the signals of Cx40 plus Cx43 look higher than Cx40 plus Cx37 in F; however, most of the spots in these two images are a mixture of red and green. All images, at the same magnification, are oriented as described in Fig 3. d, day; m, month. Bar = 15 µm.


 
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Table 1. Analysis of connexin co-localization in double-label experimentsa

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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Figure 6. Thin-section electron micrographs demonstrating typical morphology of aortic endothelium and its gap junctions at different time points in the first month of life. (A–E) Endothelium at birth. (A) At this time, endothelial cells are typically not flattened and the extracellular space between the endothelial cell and the subjacent medial smooth muscle is mainly occupied by a thin, compact layer of inhomogeneous density (arrows). (B) A complex, convoluted intercellular junctional border (arrows). Note that the adjacent cells contact closely within the junctional zone, which is different from C, where the contact is intermittent along the cell border (arrows). (D) An area where four endothelial cells crowd. (E) One large gap junction is seen at a relatively straight cell border (arrow). (Inset) High-magnification view of this gap junction with its characteristic pentalaminar structure. (F) A 7-day-old animal, showing a gap junction (arrow), the high magnification view of which is in the inset. (G) An animal 28 days old. Compared to A, the endothelial cell has become flattened, while the internal elastic lamina has become more extensive and gives a different appearance (arrows). (Inset) High-magnification view of three consecutive gap junctions (arrows) along a long, curvilinear junctional border, is from near this field. Images A–D and G are at the same magnification; images E and F are at the same magnification; all the insets are at the same magnification. Bars = 1 µm.

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 cell–cell 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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Figure 7. Thin-section electron micrographs demonstrating morphology of aortic endothelium and their gap junctions in animals 16 months of age. (A) An endothelial cell of bizarre shape, beneath which the subendothelial zone is composed of loose reticular structures and whorls of dense substances. Three gap junctions are clustered at the right border of the cell (arrows). (Inset) A high-magnification view of the largest one. (B) An octopus-like endothelial cell with its feet penetrating into the subendothelial zone. The cell processes are easily identified by the content of electron-dense inclusions (arrows). (C) In the cytoplasmic compartment near the nucleus, there is an annular gap junction (arrow), the high-magnification view of which is shown in the inset. All images except the insets are at the same magnification; all the insets are at the same magnification. Bars = 0.5 µm.


  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 (Yeh et al. 1997a ). By applying our en face immunoconfocal viewing technique in conjunction with the well-characterized, isotype-specific anti-connexin antisera in the present study, high-definition plan views of gap junction organization and distribution were visualized in individual cells over relatively large endothelial expanses, an approach that with specially designed computer software permits quantitative analysis of the expression of the component immunolabeled connexin signal as well as their relative content within the junctions (Green et al. 1993 ; Blackburn et al. 1995 ; Ko et al. 1999 ; Yeh et al. 1997b , Yeh et al. 1998 ; Yeh et al. in press ). In addition, as the borders of endothelial cells are outlined by the immunolabeled gap junction spots, the luminal surface area of individual cells can be measured. This approach therefore enables detailed examination of the interrelationship between age, cell size, and expression of the endothelial gap junctions and their connexins in situ in the animal.

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 (Blackburn et al. 1997 ), ovary (Risek et al. 1995 ; Okuma et al. 1996 ), and heart (Gourdie et al. 1992 ; Peters et al. 1994 ). In these tissues or organs, alteration of connexin expression is linked to functional maturation. For example, expression of Cx37 in the oocyte during growth is critical for reproduction, as revealed by a Cx37 gene knockout study, which showed that the gene deficient animals are infertile (Simon et al. 1997 ). In cardiac muscle, changes in connexin expression and gap junction distribution during development are implicated in alterations in anisotropic properties of working myocardium (Peters et al. 1994 ; Angst et al. 1997 ). Although we do not know whether the change of endothelial connexin expression during the first month, as shown in the present study, is critical for the integrity of endothelial cell function, reports from single connexin gene knockout studies provide some clues. In animals lacking the Cx37 or Cx40 gene, growth occurs to adulthood with a normal looking vascular system (Simon et al. 1997 , Simon et al. 1998 ; Kirchhoff et al. 1998 ). The same situation is reported in mice heterozygous (i.e., containing a single copy) for the Cx43 gene (Guerrero et al. 1997 ). These findings indicate that the animal does not necessarily require Cx37 or Cx40 protein, or dual copies of Cx43 genes, for maturation of the vascular system. Detailed examination and long term follow-up of these animals is necessary to determine if these genetically manipulated animals ultimately exhibit vascular pathology owing to the change in endothelial connexin expression. With regard to total absence of Cx43, these homozygous knockout animals fail to survive the perinatal period owing to a cardiac anomaly, although they have no apparent endothelial change (Reaume et al. 1995 ). Animals in which both Cx37 and Cx40 genes are deleted also die prenatally, but in this case there appears to be a vascular endothelial defect (Goodenough et al. 1999 ). In these cases, the possibility of examining the effects of absence of Cx43 and that of both Cx37 and Cx40 during senescence is excluded.

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 (Larson et al. 1997 ). The dynamic change of environment in vivo may contribute to this difference. For example, external to the endothelium, the remaining aortic wall experiences a great deal of remodeling, including a more than twofold increase of the caliber as shown in the present study, which leads to an increase of wall tension. At the luminal side, the endothelium is exposed to an abrupt elevation of blood pressure. These physical factors are known to affect the expression of gap junctions (Watts and Webb 1996 ; Haefliger et al. 1997 , Haefliger et al. 1999 ; Cowan et al. 1998 ; Depaola et al. 1999 ). In addition, growth factors involved in the growth of endothelium, such as bFGF and PDGF, are also modulators of the gap junctions (Pepper and Meda 1992 ; Doble and Kardami 1995 ; Hossain et al. 1998 ). Although we do not know the exact effect of each factor on individual connexins in such a complex environment, the result is that the endothelial cells are equipped with differential levels of connexins towards adulthood. Such an expression pattern, as seen at 28 days of age, is consistent with our previous reports on the same artery of young adult rats (Yeh et al. 1997a , Yeh et al. 1998 ).

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 (Xie and Hu 1994 ). However, whether such a change applies to Cx40 and Cx37, and the interrelationship between the three connexins, was not determined. The present study provides insights into these key points. In accord with the in vitro examination, we clearly demonstrate that the expression of Cx43 progressively declines as the animals grow old. In addition, the other two connexins, Cx37 and Cx40, are also downregulated at old age, although the patterns of downregulation are not the same. Furthermore, compared to 28 days of age, at 16 months the increase of Cx37 spot number and total gap junction area temporally associated with the opposite direction of Cx43 expression suggests that a compensatory mechanism exists between the two connexins. In addition, such an alteration of connexin expression during senescence may contribute to endothelial dysfunction. Senescence of endothelial cells is known to be associated with this disorder, which plays a key role in the pathogenesis of atherosclerosis (Schwartz et al. 1995 ; Vanhoutte 1997 ). Previous studies have shown that endothelial dysfunction involves a spectrum of structural and functional changes, e.g., reduction of cell membrane fluidity and purinergic receptor activity (Koga et al. 1992 ; Hashimoto et al. 1999 ). The present study suggests the possibility that changes of connexin expression, with consequent alteration of intercellular communication, may be involved in this process. Because aging is only one of the factors associated with endothelial dysfunction (Ross 1995 ), whether other factors, such as hyperlipidemia and smoking, affect endothelial connexin expression in the same manner and whether manipulation of intercellular communication can modify endothelial dysfunction and the related atherosclerotic disease require further investigation.

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.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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