Biol Cell (1990) 69, 161 - 169 © Elsevier, Paris 161 Original article Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells Irene Londofio, Moise Bendayan* Department of Anatomy, Faculty of Medicine, Universit~ de Montrdal, CP 6128 Succursale A, Montrdal, Qudbec, H3C 3J7 Canada (Received 2 February 1990; accepted 24 September 1990) Summary - Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial ceils, though with different labeling intensi- ties. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface den- sity for these structures in capillary endothelial cells (12°70)compared with those in aortic endothelial (507o) and mesothelial cells (207o). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary en- dothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalem- mal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces. albumin / aorta / protein A-gold immunocytochemistry / plasmalemmal vesicles I intercellular clefts Introduction Transport of plasma proteins across the endothelial layer and their subsequent diffusion through the interstitial space of large vessels remains a highly controversial sub- ject under continuous investigation. In a previous study [26], we have demonstrated that endogenous albumin is, in fact, unevenly distributed over the interstitial space across the rat aortic wall, with significant concentrations in the intima and adventitia. In addition, a higher interstitial-to-plasma ratio for albumin was revealed in the adventitia as compared with the intima, suggesting major differences in the albumin bulk passage from the vasa vasorum and from the aortic lumen. These differences could rely on particular endothelial lining characteristics [44], local variations in the interstitial composition across the wall [26, 40] and/or on distinct driving forces affect- ing each layer [26, 41]. Although comparative studies of endothelial characteristics in different vascular beds have been undertaken [42], there is still no consensus on the mechanism of passage of circulating proteins towards the subendothelial space [31, 40]. In the study of the endothelial permeability to circulat- ing molecules, the existence of a receptor for the transen- dothelial transport of albumin through continuous endothelia has been suggested [19, 20, 44]. Albumin- binding proteins have been demonstrated in the endothe- * Correspondence and reprints lia of several mouse capillaries and postcapillary venules [19, 20, 28], and have been partially characterized [18, 38]. In other endothelial beds such as those of arteries and veins, however, albumin transport seems to occur by fluid- phase or absorptive transcytosis, in a non-receptor medi- ated process [20, 40, 44]. Thus, the same macromolecule (albumin) seems to be transported by different mechan- isms depending on the presence of a specific receptor, and on the structural and functional particularities of the en- dothelial layer studied [35, 40, 42]. Indeed, morphologi- cal differences between endothelia, particularly concerning their thickness [9], their relative number of intracellular organeUes [35, 42], and their junctional systems have been described [21, 42]. In order to compare the intracellular distribution of en- dogenous albumin in different cellular layers, we chose the rat ascendent aorta which simultaneously includes the aor- tic endothelium, the capillary endothelium of the vasa vasorum, and the mesothelium derived from the pericardi- um. In the study of vascular permeability, perfusion of exogenous albumin tagged to different markers such as colloidal gold [19, 20, 28, 46], isotopes [1, 2, 8, 16], fluorescent tracers [24, 27], and other dyes [7, 48], has been extensively applied. The use of such exogenous tracers, though extremely valuable, is confronted by major limi- tations [3, 33, 43, 47]. The detection of endogenous albu- min through the immunocytochemical approach [5, 6, 26, 33, 36, 37], allows for the in situ localization of the cir- culating protein in steady-state conditions, circumventing problems inherent to the use of exogenous tracers. Another alternative for the detection of endogenous albumin in the