Gap Junction Localization and Connexin Expression in Cytochemically Identified Endothelial Cells of Arterial Tissue

Abstract

S U M M A R Y 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)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 (Ross 1995;Stary et al. 1994). Integration of endothelial cell functions is mediated by a variety of intercellular signaling mechanisms, including direct cell-to-cell communication via gap junctions (Larson 1988). Gap junctions are specialized cell membrane domains consisting of clusters of protein channels that link the cytoplasmic compartments of neighboring cells, forming pathways for direct exchange of ions and small molecules (for reviews see Yamasaki and

Multiple Connexin Expression in Regenerating Arterial Endothelial Gap Junctions

“…For example, the precise levels of expression of the 3 endothelial connexins depends in part on vascular site, [11][12][13][14] and the similar signal levels for the 3 connexins in uninjured rat carotid artery endothelium, distinct from the patterns previously reported in aorta, pulmonary artery, and coronary artery of the same animal, are in keeping with this finding. In a recent study examining rat aorta and its branching arteries, high levels of endothelial Cx43 gap junctions were associated with areas facing turbulent flow.…”

“…Colocalization of connexins has been reported in endothelial cells of microvessels and large vessels. 11,33 In addition, our previous work in rat aorta and pulmonary artery demonstrated that individual endothelial gap-junctional plaques contain up to 3 connexins. 13,14 The present study showed that over the regrowth of the monolayer, different connexins tend to mix within individual gap junctions.…”

“…Finally, arterial rings were cut open and mounted for en face viewing of endothelial cells. All experiments included sections of rat heart as positive controls 11 ; primary antibody was omitted in negative controls. Each secondary reagent was confirmed to be species specific by secondary antibody crossover (eg, mouse primary antibody followed by anti-rabbit or anti-guinea pig secondary antibody).…”

Differential Expression of Connexin43 and Desmin Defines Two Subpopulations of Medial Smooth Muscle Cells in the Human Internal Mammary Artery

“…The specificity of these antibodies has been demonstrated by western blotting and immunolabeling of HeLa cell transfectants, and by immunogold labeling (thin sections and/or freeze-fracture cytochemistry). 7,25 8 Macrophages were detected by a mouse anti-CD68 monoclonal antibody (1:50; Dako, catalog No. M0876), and T lymphocytes by a mouse anti-CD3 monoclonal antibody (1:200; Chemicon, catalog No.…”

“…[5][6][7] In the arterial wall, endothelial cells and SMCs have distinctive but overlapping connexin expression profiles. 5,6 Although Cx40 and Cx37, and in some instances also Cx43, are coexpressed in the endothelium, 8 SMCs in general show a simpler connexin profile, predominantly expressing Cx43. 8,9 Cx43 is abundantly expressed in the media of elastic arteries, such as the aorta 10,11 and carotid artery, 3 but only at low, barely detectable or undetectable levels in muscular arteries, such as the coronary artery 12,13 and arterioles.…”