Association of interendothelial junctional 'stigmata' with areas of increased Evans blue dye-albumin uptake in the porcine thoracic aorta

Arterial endothelium exposed to silver nitrate exhibits a mesh-like pattern of lines when viewed en face. These lines have been shown to demarcate the interendothelial junction, and are often punctuated by dot-like features, referred to as “stigmata”. Evidence in the microvasculature suggests that these structures may be involved in the transendothelial transport of macromolecules, but the extent of this involvement in large arteries has not been resolved. The purpose of this work was to (1) determine to what extent these stigmata are associated with locally increased transendothelial transport of the EBD-albumin macromolecule in large arteries, and (2) identify ultrastructural changes in the endothelial barrier that are associated with stigmata.; In vivo experiments were carried out in swine to quantify EBD-albumin uptake and stigmata morphometric parameters in the thoracic aorta. The aortae were exposed to a known concentration of EBD-albumin, and subsequently stained with silver nitrate, and pressure fixed in situ. The lumenal surfaces of the aortae were then examined en face under epi-illumination, and digital images of the surface were collected at carefully selected locations along the lengths of the aortae. These digital images were then processed to quantify local EBD-albumin uptake, and stigmata morphometric parameters, including the stigmata number density and area fraction. Linear regression analysis was then performed on these results to evaluate the level of correlation between these variables. Additional experiments were performed to examine the ultrastructure of stigmata using scanning electron microscopy.; The results of these studies showed positive correlations between EBD-albumin uptake rate and stigmata morphologic parameters for each of three separate experiments. These findings are important because they are the first to demonstrate this relationship in a large artery. Further, these findings are the first to establish the quantitative relationship between macromolecular permeability and the physical number and size of stigmata in any portion of the vasculature. The efforts made toward the elucidation of stigmata ultrastructure suggested that at least the largest stigmata may demarcate locations where gaps are present in the interendothelial junctions. Ultimately, all of these results may contribute to the identification of key physical pathways for macromolecular transport across the endothelial barrier.