ARTICLE |
Correspondence to: Nicholas J. Severs, Cardiac Medicine, Imperial College School of Medicine at National Heart and Lung Institute, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vascular endothelial cells interact with one another via gap junctions, but information on the precise connexin make-up of endothelial gap junctions in intact arterial tissue is limited. One factor contributing to this lack of information is that standard immunocytochemical methodologies applied to arterial sections do not readily permit unequivocal localization of connexin immunolabeling to endothelium. Here we introduce a method for multiple labeling with specific endothelial cell markers and one or more connexin-specific antibodies which overcomes this limitation. Applying this method to localize connexins 43, 40, and 37 by confocal microscopy, we show that the three connexin types have quite distinctive labeling patterns in different vessels. Whereas endothelial cells of rat aorta and coronary artery characteristically show extensive, prominent connexin40, and heterogeneous scattered connexin37, the former, unlike the latter, also has abundant connexin43. The relative lack of connexin43 in coronary artery endothelium was confirmed in both rat and human using three alternative antibodies. In the aorta, connexins43 and 40 commonly co-localize to the same junctional plaque. Even within a given type of endothelium, zonal variation in connexin expression was apparent. In rat endocardium, a zone just below the mitral valve region is marked by expression of greater quantities of connexin43 than surrounding areas. These results are consistent with the idea that differential expression of connexins may contribute to modulation of endothelial gap junction function in different segments and subzones of the arterial system. (J Histochem Cytochem 45:539-550, 1997)
Key Words: gap junctions, endothelium, connexins, artery, heart, rat, human, confocal microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The vascular endothelium forms a continuous monolayer lining the luminal surface of the entire cardiovascular system, providing the structural and metabolic interface between the blood and underlying tissues. Endothelial integrity is essential for maintenance of healthy tissue function, and perturbations of endothelial structure and function are critical to the pathogenesis of vascular disease (
Information to date on the expression of connexins in endothelial cells has relied largely on the use of cultured cells and on Northern and Western blotting of endothelial scrapings. In situ immunocytochemical localization studies of the intact vessel have thus far been largely restricted to the microvasculature (e.g.,
Investigation of connexin expression in the endothelium of the intact vessel therefore requires simultaneous visualization of unequivocally identified endothelial cells with immunolabeled gap junctions. To this end, the present study set out to develop a method for multiple labeling with specific endothelial cell markers and one or more connexin-specific antibodies. Application of this method has made it possible to initiate investigation of the distribution of gap junctions and the diversity of connexin expression in endothelial cells in the intact tissue in a range of arterial vessel types.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Preparation
Specimens of aorta, coronary artery, and endocardium were obtained from adult male Sprague-Dawley rats (318-450 g). One series of five animals was sacrificed by dislocation of the neck and the arterial samples were rapidly washed with PBS containing heparin (10 U/ml), cut into 5-mm-thick segments, and frozen immediately without fixation. A second series of six animals was perfusion-fixed in paraformaldehyde before freezing. These animals were anesthetized by IP injection of midalozam-hypnorm and perfused retrogradely, via a catheter in the abdominal aorta, with heparinized PBS, followed by phosphate-buffered 2% paraformaldehyde (pH 7.4) for 20 min. Five-mm-thick transverse rings of fixed arterial tissues were incubated in 30% sucrose in PBS for 1 hr before freezing. Preparation of rat tissues was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.
Human coronary arteries were also studied. Samples of undiseased coronary artery were obtained from the explanted hearts of five male patients (mean age 45.3 ± 6.7 years) with endstage dilated cardiomyopathy. After ischemic arrest and removal of the heart, segments of coronary artery were immediately dissected out in the operating theater and placed in 2% paraformaldehyde (phosphate-buffered) for 1 hr, followed by PBS wash. Up to nine segments from different coronary arteries were obtained per heart. From each segment, one 5-mm-thick transverse arterial ring was selected for study. Work on human tissues was conducted according to institutional ethical committee policies.
All samples were swiftly immersed in isopentane held close to its freezing point (-160C) using liquid nitrogen, and subsequently stored under liquid nitrogen before cryosectioning. Ten-µm-thick cryosections of the arterial rings were cut transversely and obliquely using a cryostat. Oblique cutting provided extended tangential sections in which portions of endothelium were viewed en face.
Immunofluorescence Labeling of Gap Junctional Connexins and Endothelial Cell Identification
Anti-connexin Antibodies.
Three principal antibodies were used for the immunofluorescence detection of the gap junctional proteins, connexins37, 40, and 43. Those against connexins37 and 40 were polyclonal antisera raised in rabbits. The connexin37 antiserum was raised against a synthetic peptide corresponding to residues 266-281 ("D37") of the cytoplasmic C-terminal tail of the rat connexin37 (a gift from P. Meda) (
Endothelial Cell Markers. Three endothelial cell markers were used, as follows. The lectins Ulex europaeus agglutinin 1 (UEA1) conjugated with FITC, and Bandeiraea simplicifolia isolectin B4 (BS1) conjugated with FITC, both purchased from Sigma (Dorset, UK), were used to label endothelial cells of human and rat arteries, respectively. As an alternative, rabbit anti-human Von Willebrand factor (anti-VWF) polyclonal antiserum (Dako; Wycombe, UK) was used to label endothelial cells of both rat and human specimens.
Secondary Antibody/Detection Systems. For standard connexin labeling, both in single labeling experiments and in combination with endothelial marking (see below), the secondary antibody/detection systems used were biotinylated sheep anti-rabbit or biotinylated sheep anti-mouse immunoglobulins with Texas Red-streptavidin (Amersham Life Science; Poole, UK). For double labeling of two connexin isoforms (both with and without endothelial marking), we used donkey anti-mouse immunoglobulin conjugated to CY5 and donkey anti-rabbit immunoglobulin conjugated to CY3 (both from Chemicon). Secondary antibody/detection systems used for endothelial marking were as follows: (a) rabbit anti-fluorescein isothiocyanate immunoglobulin (anti-FITC; Dako); (b) swine anti-rabbit-fluorescein isothiocyanate (anti-rabbit-FITC; Dako). Reagents a and b were used to amplify the signal associated with lectin. Reagent b alone was used for labeling of Von Willebrand factor.
Combined Labeling of Gap Junctions with Endothelial Marking. Sections were mounted on poly-L-lysine-coated slides. After overnight air-drying in the -20C freezer, the unfixed rat sections were first immersed in -20C methanol for 5 min, after which all sections were rinsed in PBS for 5 min and treated with 0.1% Triton X-100 in PBS for 15 min. This was followed by blocking in PBS containing 0.5% bovine serum albumin for 15 min and incubation with the anti-connexin antibody of choice. Different conditions were found to be optimal for each of the antibodies, as follows: connexin37 antibody (dilution 1:300) at room temperature (RT) overnight; connexin40 antibody (dilution 1:1000) at 37C for 30 min; connexin43 monoclonal antibody (dilution 1:1000) at RT overnight. Sections were then treated for 1 hr at RT with biotinylated sheep anti-rabbit (for connexins37 and 40) or biotinylated sheep anti-mouse (for connexin43) at a concentration of 1:250, with subsequent fluorescent visualization using Texas Red-streptavidin (1:250). In experiments in which two connexins were simultaneously localized in the same section, sequential incubation with each of the anti-connexin antibodies was followed by incubation at RT for 1 hr with a mixture of the two secondary antibodies (CY3 and CY5; 1:250). This approach was feasible for the combination of connexins 40 and 43.
For simultaneous marking of endothelial cells, one of two alternative procedures was used: lectin labeling or localization of VWF. For the former, the connexin immunolabeled sections were exposed either to lectin BS1-FITC (5 µg/ml in PBS) at RT for 2 hr (for rat specimens) or lectin UEA1-FITC (1 µg/ml in PBS) for 1 hr (for human specimens). These sections were then incubated for 1 hr at RT in rabbit anti-FITC (dilution 1:500), followed by swine anti-rabbit-FITC (dilution 1:25) for 1 hr at RT to enhance fluorescent visualization. For the VWF method, the sections were treated for 1 hr at RT with anti-VWF (dilution 1:500), followed by swine anti-rabbit-FITC (dilution 1:25). After washing in PBS, the slides were mounted with Citifluor mounting medium (Agar; Essex, UK). The sections were given a thorough wash in PBS between each step. The endothelial marking approach was successfully applied to sections that had been prelabeled for one or for two connexin types.
Corresponding experiments were conducted in which each of the anti-connexin antibodies was applied alone (i.e., no endothelial marker) and the endothelial marker used alone (i.e., no connexin labeling). As positive controls, gap junction labeling in the medial smooth muscle cells (connexin43 antibody) and in the endothelial cells of vasa vasorum (connexin40 and 37 antisera) was used. Negative controls included omission of primary antibody and peptide inhibition. In addition, each secondary reagent was confirmed to be species-specific by secondary antibody crossover (i.e., mouse primary antibody followed by anti-rabbit secondary; rabbit primary antibody followed by anti-mouse secondary).
Confocal Laser Scanning Microscopy and Correlative Histology
Immunolabeled sections were examined by confocal laser scanning microscopy using a Leica TCS 4D, equipped with an argon/krypton laser and fitted with the appropriate filter blocks for detection of fluorescein, Texas Red, and CY3 and CY5 fluorescence. The images were taken using simultaneous dual or triple channel scanning and transformed into projection views using sets of five consecutive single optical sections taken at 1-µm intervals. All specimens were examined within 24 hr of immunolabeling. Adjacent sections to those used for immunolabeling, stained with hematoxylin and eosin, were examined using standard brightfield optics for comparative histological examination.
Comparison of the immunoconfocal results obtained using (a) methanol-fixed cryosections of directly frozen specimens and (b) cryosections of standard (20-min) paraformaldehyde-fixed specimens of rat arteries revealed identical connexin labeling patterns, with no differences in detectability between the preparative procedures. However, we found that paraformaldehyde fixation exceeding 30 min led to a fixation time-dependent decrease in connexin detectability.
For comparison of the distribution and relative quantities of the immunofluorescence signal for each connexin type in the different vessels, 10 sections were taken from each of six samples for each rat arterial type or, for human coronary artery, from five separate samples. Each sample came from a different animal or patient. The sections were assessed visually at a magnification of x 400 for the distribution of connexin immunofluorescence and were scored for the relative amount of signal by estimating the percentage of endothelial area (as revealed with the cell marker) that exhibited distinct punctate connexin labeling delineating the cell borders. With this system, the maximum score of 100% (+++++) was applied where punctate label was apparent around the borders of all endothelial cells observed, and the minimal score of <1% to instances in which less than 1% of the endothelial area observed included positive connexin signal at the cell borders. Intermediate scores between these extremes are as defined in the footnote to Table 1. It should be noted that differences in signal intensity in immunolabeling experiments do not necessarily directly reflect relative abundance of different connexin types. Other factors relating to the distinctive properties of different antibodies (e.g., affinity for their respective antigens, sensitivity to processing protocols) are also involved. In the present study, however, the distribution patterns and extent of labeling for connexin37 were reproducibly observed with two separate antibodies and those for connexin43 with three separate antibodies.
|
Postembedding Immunogold Labeling for Electron Microscopy
To verify results obtained from double labeling for connexins 40 and 43 at the electron microscopic level, we carried out immunogold labeling of sections of rat aorta that had been embedded at low temperature in Lowicryl K4M (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In arterial sections conventionally stained with hematoxylin and eosin, endothelial cells are typically viewed in a variety of orientations, ranging from transverse to oblique (Figure 1). In sections immunolabeled with anti-connexin antibodies, the limits of the endothelium could not always be discerned with confidence, even with optimized correlative phase-contrast examination and, consequently, attributing positive or negative signal at the luminal surface to endothelium was problematic. Figures 2-5, taken from rat aorta, illustrate this problem and how it can be effectively overcome using endothelial cell markers.
|
|
Figures 2A and 3A illustrate typical patterns of connexin43 labeling observed in the absence of endothelial marker. In the former the labeling increases in abundance in a band towards the luminal side, and in the latter uniform labeling is observed extending to the luminal edge. By simultaneously marking the endothelium with anti-VWF (Figure 2B), it becomes clear that the band of increased labeling in Figure 2A coincides precisely with the presence of tangentially cut endothelium (as depicted in Figure 1A). The absence of a similar band of elevated connexin43 labeling in Figure 3A might be attributed to loss of the endothelium. However, application of the endothelial marker demonstrates a narrower band of tangentially cut endothelium, which in this orientation reveals connexin43 gap junctions at a density similar to those of the underlying smooth muscle cells (Figure 3B). A comparable connexin43 gap junction pattern is apparent where the endothelium is viewed in precise transverse section (Figure 4), equivalent to the plane shown in Figure 1B.
By applying antibodies against other connexin types, we demonstrated that, apart from connexin43 (Figures 2-4), rat aortic endothelial cells express connexins 40 (Figure 5) and 37 (Figure 6). The examples in Figure 5 further illustrate how detachment of the endothelium during processing may, in the absence of endothelial marking, confound interpretation of connexin immunolabeling results, and that Bandeiraea simplicifolia isolectin B4 (Figure 5A) produces comparable results to anti-VWF (Figure 5B) when used as the endothelial marker. Comparison of the results obtained with antibodies against the three different connexin types revealed distinctive immunolabeling patterns in rat aortic endothelial cells. Whereas connexin43 signal was abundant in both the endothelium and medial smooth muscle cells, connexin40 was confined to the endothelium (Figures 2-5). Both connexin43 and connexin40 labeling revealed prominent punctate patterns delineating individual endothelial cells in en face views. Overall, the extent and intensity of connexin40 signal exceeded those of connexin43. Connexin37 differed from connexins 43 and 40 by showing a markedly heterogeneous distribution (Figure 6). Although focal areas of rat aortic endothelium revealed small but clearly resolved connexin37 label at the cells' borders (Figure 6A), major stretches of the neighboring endothelium revealed no connexin37 label (Figure 6B).
Comparison of different vessels revealed characteristic and distinctive patterns of endothelial connexin expression in each. For example, the endothelium of the rat ventricular endocardium just below the mitral valve region expressed only connexin43 (Figure 7), whereas elsewhere in the ventricular endocardium some connexin40 and 37 was also detectable, albeit in small quantities. Connexins40 and 37 were widespread in the atrial endocardial endothelium. In rat coronary arteries, connexin40 was expressed abundantly (Figure 8) whereas connexin43 was typically undetectable except in a zone close to its junction with the aorta. Connexin37 was present in the coronary arteries but showed a heterogeneous pattern of distribution similar to that of the aorta (not illustrated). For all vessels, the extent and intensity of the labeling patterns observed for connexins 37 and 43 were identical when alternative antibodies were applied. Table 1 summarizes the connexin distribution characteristics for the vessels examined.
|
To examine whether the differences observed were a vessel-specific or species-specific feature, the pattern of connexins in rat coronary artery was compared with that in human coronary artery. In human coronary arterial endothelium, as in the rat, connexin40 was the most abundant connexin (Figure 9A) and connexin37 was heterogeneously distributed (Figure 9B). Connexin43 was virtually absent, although close inspection revealed very occasional small spots (Figure 9C). Histological assessment confirmed absence of atherosclerotic lesions in the human coronary artery specimens examined.
To determine the relationships between the distributions of two connexin types, experiments were conducted in which connexins 43 and 40 were simultaneously localized in cytochemically identified endothelial cells (Figure 7 and Figure 8). Application of this technique to the endocardial endothelium and to the coronary endothelium emphasized differential expression of connexins in closely adjacent compartments comprising different cell types. In the endocardium, the connexin43-expressing endothelial cells were seen adjacent to myocytes of the left bundle branch of the atrioventricular conduction system which, as reported previously (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study has demonstrated the feasibility of simultaneously labeling endothelial cells with cell-specific markers and localizing one or more connexins in sections of the intact arterial wall. Such a strategy facilitates localizing precisely where different connexins are expressed and how gap junctions of different connexin content are organized in the arterial endothelium in situ.
Our comparison of connexin immunolabeling results with and without simultaneous visualization of the endothelium demonstrates the variety of interpretative difficulties that arise in the absence of cell marking. These difficulties stem from two principal features of the endothelium. First, endothelial cells in the normal artery are thin and flat, forming an inconspicuous monolayer along the inner interface of the vascular wall. Second, the endothelium is readily detached from the vascular wall during the course of processing and cryosectioning, and endothelial denudation is a common feature of vascular pathologies. Therefore, positive connexin labeling along the luminal interface does not necessarily represent that of endothelial cells. Conversely, lack of immunolabeling for a given connexin at the luminal surface may be due either to absence of the connexin or to absence of endothelial cells. Portions of endothelium are frequently tangentially sectioned, even in transversely cut arterial rings, so that the boundary between endothelium and the underlying medial layer in sectional views varies considerably in depth. Furthermore, because two of the connexins reported to be expressed in endothelial cells, connexin43 and connexin40, have also been reported in vascular smooth muscle cells (
The basic strategy of using two fluorochromes that emit different wavelength spectra [e.g., one green (FITC) with one red fluorochrome (e.g., Texas Red)], applied here to achieve simultaneous endothelial marking and connexin localization, is widely used in cytochemistry. Extending this principle, we used up to three fluorochromes in the present study in the following combinations: (a) FITC (endothelium) plus Texas Red (connexin); (b) FITC (endothelium) plus CY3 and CY5 (two distinct connexins). In the vascular wall, elastic laminae give strong autofluorescence, which tends to mask FITC fluorescence, and for this reason FITC labeling was unsuitable for labeling gap junctions in the vessel wall. Even when used as the endothelial marker, FITC fluorescence in lectin-labeled specimens was difficult to distinguish from the internal elastic lamina which, in rat arteries, lies immediately adjacent to the endothelium. This problem was effectively overcome by amplifying the signal using unlabeled rabbit anti-FITC antibodies followed by anti-rabbit-FITC. The signal achieved with anti-VWF antibody followed by FITC secondary had the advantage of being sufficiently strong without amplification. Because of its simplicity, the VWF marker is our method of choice and should be applicable to endothelia of most vessels. However, because the quantity of VWF expressed in the endothelia of different vessels is reported to vary (
Earlier ultrastructural studies, especially those applying freeze-fracture electron microscopy, have provided a comprehensive picture of the organization, size, and distribution of endothelial gap junctions in situ (
An important finding to emerge from the present study is that complementary investigation of the three connexin types reveals quite distinctive labeling patterns in different vessels. For example, whereas aortic endothelial cells have extensive, prominent connexin40 and 43 with heterogeneously distributed connexin37 labeling, coronary artery endothelium, although showing similar features for connexin40 and 37, reveals a lack of connexin43 labeling. The reproducibility of these labeling patterns with alternative probes to the same connexin type suggests that these distinctive features reflect true underlying differences in the relative abundance of the three connexins, rather than being due to technical factors such as differences in antibody affinities or epitope accessibilities. The presence of three connexin types with distinct immunolabeling patterns in endothelia of different arteries has potentially important functional implications. Recent experimental studies on cells stably transfected with cDNAs encoding different connexins indicates that the properties of gap junction channels (e.g., unitary conductance, voltage sensitivity, molecular permeability, and ionic selectivity) vary according to the specific connexin expressed (
Our experiments on simultaneous localization of connexins40 and 43 by immunoconfocal and immunoelectron microscopy indicate that, in rat aortic endothelium, although a few junctions may contain one or other of these connexins, the major population of junctions contains a mixture of the two. A similar co-localization has recently been reported by confocal microscopy in microvascular (arteriolar) endothelium from hamster cheek pouch (
In conclusion, the present findings demonstrate that segmental differentiation of vascular endothelium extends to differences in the distribution patterns and relative abundances of the specific types of gap junctional connexins expressed. Such differential expression of connexins may contribute to modulation of gap junction function in different segments of the arterial wall. The combined endothelial cell marking/connexin labeling approach for confocal microscopy introduced here will enable further detailed, reliable investigation of the diversity of endothelial connexin expression.
![]() |
Acknowledgments |
---|
Supported in part by project grants from the British Heart Foundation (grant no. PG 93136) and the Wellcome Trust (grant no. 046218/Z/95).
We wish to thank all colleagues who contributed gifts of antibodies or participated in their production, in particular Dr Colin Green and Dr Robert Gourdie (anti-connexin43 "HJ" and anti-connexin40). The anti-connexin37 antibody used here was produced at the University of Geneva Medical School, and we thank Prof Paolo Meda and Dr J-A. Haefliger (DMIB, Lausanne) for this gift.
Received for publication June 19, 1996; accepted November 14, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bastide B, Neyses L, Ganten D, Paul M, Willecke K, Traub O (1993) Gap junction protein connexin40 is preferentially expressed in vascular endothelium and conductive bundles of rat myocardium and is increased under hypertensive conditions. Circ Res 73:1138-1149[Abstract]
Beblo DA, Wang HZ, Beyer EC, Westphale EM, Veenstra RD (1995) Unique conductance, gating, and selective permeability properties of gap junction channels formed by connexin40. Circ Res 77:813-822
Beyer EC (1993) Gap junctions. Int Rev Cytol 137C:1-37
Beyer EC, Paul DL, Goodenough DA (1987) Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621-2629[Abstract]
Beyer EC, Reed KE, Westphale EM, Kanter HL, Larson DM (1992) Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle. J Membr Biol 127:69-76[Medline]
Blackburn JP, Peters NS, Yeh H-I, Rothery S, Green CR, Severs NJ (1995) Upregulation of connexin43 gap junctions during early stages of human coronary atherosclerosis. Arterioscler Thromb Vasc Biol 15:1219-1228
Bruzzone R, Haefliger J-A, Gimlich RL, Paul DL (1993) Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell 4:7-19[Abstract]
Bruzzone R, White TW, Paul DL (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 238:1-27[Abstract]
Carlemalm E, Garavito RM, Villiger W (1981) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126:123-129
Carter TD, Chen XY, Carlile G, Kalapothakis E, Ogden D, Evans WH (1996) Porcine aortic endothelial gap junctions: Identification and permeation by caged InsP3. J Cell Sci 109:1765-1773
Chanson M, Spray DC (1995) Electrophysiology of gap junction conductance. In Huizinga JD, ed. Pacemaker Activity and Intercellular Communication. Boca Raton, FL, CRC Press, 51-72
Darrow BJ, Laing JG, Lampe PD, Saffitz JE, Beyer EC (1995) Expression of multiple connexins in cultured neonatal rat ventricular myocytes. Circ Res 76:381-387
El Aoumari A, Fromaget C, Dupont E, Reggio H, Durbec P, Briand J-P, Böller K, Kreitman B, Gros D (1990) Conservation of a cytoplasmic carboxy-terminal domain of connexin43, a gap-junctional protein, in mammalian heart and brain. J Membr Biol 115:229-240[Medline]
Elfgang C, Eckert R, Lichtenberg-Fraté H, Butterweck A, Traub O, Klein RA, Hülser DF, Willecke K (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 129:805-817[Abstract]
Gebrane-Younès J, Drouet L, Caen JP, Orcel L (1991) Heterogeneous distribution of Weibel-Palade bodies and Von Willebrand factor along the porcine vascular tree. Am J Pathol 139:1471-1484[Abstract]
Gilula NB (1992) Gap junctions and intercellular communication. Semin Cell Biol 3:1-91
Gourdie RG (1995) Microscopy of intercellular communicating junctions. Microsc Res Tech 31:337-468
Gourdie RG, Green CR, Severs NJ, Anderson RH, Thompson RP (1993) Evidence for a distinct gap-junctional phenotype in ventricular conduction tissue of the developing and mature avian heart. Circ Res 72:278-289[Abstract]
Green CR, Severs NJ (1993) Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease. Histochemistry 99:105-120[Medline]
Gros D, Jarry-Guichard T, ten Velde I, De Mazière AMGL, Van Kempen MJA, Davoust J, Briand JP, Moorman AFM, Jongsma HJ (1994) Restricted distribution of connexin40, a gap junctional protein, in mammalian heart. Circ Res 74:839-851[Abstract]
Harfst E, Severs NJ, Green CR (1990) Cardiac myocyte gap junctions: evidence for a major connexon protein with an apparent relative molecular mass of 70,000. J Cell Sci 96:591-604[Abstract]
Hüttner I (1985) Aortic endothelial cell during regeneration: remodeling of cell junctions, stress fibers, and stress fiber-membrane attachment domains. Lab Invest 53:287-302[Medline]
Hüttner I, Peters H (1978) Heterogeneity of cell junctions in rat aortic endothelium: a freeze-fracture study. J Ultrastruct Res 64:303-308[Medline]
Kanter HL, Laing JG, Beau SL, Beyer EC, Saffitz JE (1993) Distinct patterns of connexin expression in canine Purkinje fibers and ventricular muscle. Circ Res 72:1124-1131[Abstract]
Kumar NM, Gilula NB (1996) The gap junction communication channel. Cell 84:381-388[Medline]
Larson DM (1988) Intercellular junctions and junctional transfer in the blood vessel wall. In Ryan U, ed. Endothelial Cells III. Boca Raton, FL, CRC Press, 75-88
Larson DM, Haudenschild CC, Beyer EC (1990) Gap junction messenger RNA expression by vascular wall cells. Circ Res 66:1074-1080[Abstract]
Little TL, Beyer EC, Duling BR (1995a) Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am J Physiol 268:H729-H739
Little TL, Xia J, Duling BR (1995b) Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall. Circ Res 76:498-504
Meda P, Vozzi C, Ullrich S, Dupont E, Charollais A, Sutter E, Bosco D (1995) Gland cell connexins. In Kanno Y, Kataoka K, eds. Progress in Cell Research. Vol 4. New York, Elsevier Science, 281-287
Moore LK, Burt JM (1995) Gap junction function in vascular smooth muscle: influence of serotonin. Am J Physiol 269:H1481-H1489
Moore LK, Burt JM (1994) Selective block of gap junction channel expression with connexin-specific antisense oligodeoxynucleotides. Am J Physiol 267:C1371-C1380
Moreno AP, Rook MB, Fishman GI, Spray DC (1994) Gap junction channels: distinct voltage-sensitive and -insensitive conductance states. Biophys J 67:113-119[Abstract]
Mukai K, Rosai J, Burgdorf WHC (1980) Localization of factor VIII-related antigen in vascular endothelial cells using an immunoperoxidase method. Am J Surg Pathol 4:273-276[Medline]
Pepper MS, Montesano R, El Aoumari A, Gros D, Orci L, Meda P (1992) Coupling and connexin 43 expression in microvascular and large vessel endothelial cells. Am J Physiol 262:C1246-C1257
Reed KE, Westphale EM, Larson DM, Wang H-Z, Veenstra RD, Beyer EC (1993) Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein. J Clin Invest 91:997-1004[Medline]
Rennick RE, Connat J-L, Burnstock G, Rothery S, Severs NJ, Green CR (1993) Expression of connexin43 gap junctions between cultured vascular smooth muscle cells is dependent upon phenotype. Cell Tissue Res 271:323-332[Medline]
Ross R (1995) Cell biology of atherosclerosis. Annu Rev Physiol 57:791-804[Medline]
Schneeberger EE (1981) Segmental differentiation of endothelial intercellular junctions in intra-acinar arteries and veins of the rat lung. Circ Res 49:1102-1111[Medline]
Schwartz SM, Benditt EP (1973) Cell replication in the aortic endothelium: a new method for study of the problem. Lab Invest 28:699-707[Medline]
Sehested M, Hou-Jensen K (1981) Factor VIII related antigen as an endothelial cell marker in benign and malignant disease. Virchows Arch [A] 391:217-225
Severs NJ (1989) Constituent cells of the heart and isolated cell models in cardiovascular research. In Piper HM, Isenberg G, eds. Isolated Adult Cardiomyocytes. Vol 1. Boca Raton, FL, CRC Press, 3-41
Simionescu N, Simionescu M (1988) The cardiovascular system. In Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology. Munich, Urban & Schwarzenberg, 355-402
Simionescu M, Simionescu N, Palade GE (1976) Segmental differentiations of cell junctions in the vascular endothelium. Arteries and veins. J Cell Biol 68:705-723[Abstract]
Simionescu M, Simionescu N, Palade GE (1975) Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature. J Cell Biol 67:863-885[Abstract]
Slot JW, Geuze HJ (1984) Gold markers for single and double labeling of ultrathin cryosections. In Polak JM, Varndell IM, eds. Immunolabelling for Electron Microscopy. Amsterdam, Elsevier Science Publishers, 129-142
Sosinsky G (1995) Mixing of connexins in gap junction membrane channels. Proc Natl Acad Sci USA 92:9210-9214[Abstract]
Stary HC, Bleakley Chandler A, Glagov S, Guyton JR, Insull W, Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW (1994) A definition of initial, fatty streak, and intermediate lesions of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb 14:840-856[Abstract]
Stephenson TJ, Griffiths DWR, Mills PM (1986) Comparison of Ulex europaeus I lectin binding and factor VIII-related antigen as markers of vascular endothelium in follicular carcinoma of the thyroid. Histopathology 10:251-260[Medline]
Veenstra RD, Wang HZ, Beblo DA, Chilton MG, Harris AL, Beyer EC, Brink PR (1995) Selectivity of connexin-specific gap junctions does not correlate with channel conductance. Circ Res 77:1156-1165
Wharton J, Gordon L, Power RF, Polak JM (1990) Microscopic method of investigating endothelium. In Warren JB, ed. The Endothelium: An Introduction to Current Research. Chichester, Wiley-Liss, 253-261
White TW, Paul DL, Goodenough DA, Bruzzone R (1995) Functional analysis of selective interactions among rodent connexins. Mol Biol Cell 6:459-470[Abstract]
Willecke K, Heynkes R, Dahl E, Stutenkemper R, Hennemann H, Jungbluth S, Suchyna T, Nicholson BJ (1991) Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung. J Cell Biol 114:1049-1057[Abstract]
Wolburg H, Rohlmann A (1995) Structure-function relationships in gap junctions. Int Rev Cytol 157:315-356[Medline]
Xie H, Hu VW (1994) Modulation of gap junctions in senescent endothelial cells. Exp Cell Res 214:172-176[Medline]
Yamasaki H, Naus CCG (1996) Role of connexin genes in growth control. Carcinogenesis 17:1199-1213[Medline]