Transendothelial transport of serum albumin: a quantitative immunocytochemical study

Abstract

Abstract. The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody: Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, ptasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68 % of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNPtagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.central point of interest in the biology of the cardiovascular system concerns the transendothelial transport (transcytosis) of plasma molecules. A large amount of data was generated by experiments in which radioactively tagged molecules of different sizes and physiological significance have been injected or perfused in segments of the body or in isolated organs. The vascular permeability was evaluated by measuring the radioactivity collected in lymph, retained in the tissue, or cleared from the circulating fluids (42). The interpretation of these data suggested some models in which the endothelial layer was considered as a passive sieve endowed with pores of different dimensions. Although widely accepted by physiologists, these models, based solely on diffusional and convectional processes, did not completely explain the experimental data regarding the vascular permeability to macromolecules, leaving room for other possible mechanisms of endothelial transport (35). Purely conceptual, these models prompted a considerable number of morphological studies on vascular endothelium in an attempt to directly visualize the transendothelial pathways followed by macromolecules. However, the putative endothelial pore system has no...

Alterations of the Rat Mesentery Vasculature in Experimental Diabetes

“…DNP attached to BSA allowed us to immunocytochemically localize the tracer with high resolution, at the ultrastructural level, in compartments relevant for the process of transcytosis. The quantitative relationship between the density of colloidal gold immunolabeling and the actual concentration of the detected molecule has been demonstrated (Ghitescu and Bendayan 1992;Griffith and Hoppeler, 1986;Slot et al, 1989) and is generally accepted. At 15 minutes circulation time, the tracer is still far from reaching an equilibrium between the vascular lumen and the interstitium (Ghitescu, Bendayan, 1992).…”

“…Crystallized BSA was purchased from Sigma Chemical, St. Louis, Missouri; 2,4 dinitrobenzene sulfonic acid was from Aldrich Chemical, Milwaukee, Wisconsin; Na 125 I, carrier free, was from ICN Biochemicals, Missisauga, Ontario, Canada; and chloramine T immobilized on polystyrene beads (Iodo-Beads) was from Pierce Chemical, Rockford, Illinois. The tracer preparation procedure was similar to that described previously (Ghitescu and Bendayan, 1992). In brief, BSA was tagged with DNP according to the method of Little and Eisen (1967).…”

“…The quantitative relationship between the density of colloidal gold immunolabeling and the actual concentration of the detected molecule has been demonstrated (Ghitescu and Bendayan 1992;Griffith and Hoppeler, 1986;Slot et al, 1989) and is generally accepted. At 15 minutes circulation time, the tracer is still far from reaching an equilibrium between the vascular lumen and the interstitium (Ghitescu, Bendayan, 1992). Because of this large transendothelial gradient, the putative 125 I-DNP-BSA flux through the interendothelial junctions would be at its maximal value and therefore easily detected as a large population of haptenated molecules in transit through the junctional space.…”

“…These alterations are reported as increased permeability to macromolecules, vascular hyperplasy, and angiogenesis. To demonstrate the changes in permeability, diabetic and normal rats were injected iv with a double-tagged tracer, 125 I-DNP-BSA, whose fate in circulation has been previously shown to be virtually identical to that of the native, endogenous serum albumin (Ghitescu and Bendayan, 1992;Kitteringham et al, 1985). This experimental design preserved all of the essential conditions (presence of plasma proteins, blood cells, and normal hemodynamic factors) for an unbiased assessment of the vascular permeability under physiological conditions.…”

The Neutrophil-Activating Protein of Helicobacter pylori Crosses Endothelia to Promote Neutrophil Adhesion In Vivo

“…For this purpose, we monitored the passage of FITC-BSA, applied in the upper chamber, through endothelial monolayers in the presence of HP-NAP. The transport of native albumin across continuous endothelium occurs via noncoated plasmalemmal vesicles or caveolae rather than via the paracellular route (34,35), making BSA a good marker for the evaluation of endothelial integrity (29). As shown in Fig.…”