Distribution of anionic sites on microvascular endothelium of the chick chorioallantoic membrane

It is generally accepted that luminal surfaces of adult microvascular endothelia present an anionic barrier that limits passage of anionic macromolecules. To assess the ontogeny of the barrier, temporal and spatial expression of endothelial anionic sites was evaluated in the chorioallantoic membrane of chicken embryos from days 4.5 to 18 of incubation. After an initial flush, the vessels were perfused with cationic ferritin (CF, 1.0 mg/ml in PBS) for 2 min. Following a second flush to remove unbound CF, the chick chorioallontoic membranes (CAMs) were fixed and processed for electron microscopy. Continuous CF binding was revealed on the luminal endothelium, the junctional clefts and the plasmalemmal vesicles from days 4.5 to 14. However, by day 18, anionic sites had become discontinuous. Prior perfusion with protamine sulfate abolished CF binding and facilitated native ferritin binding. Further ultrastructural evaluation, using peroxidase labeled LFA lectin, revealed sialic acid moieties in patches on the CAM endothelium. Thus, in early chick embryogenesis, the CAM endothelium displays a continuous pattern of luminal anionic sites comprised in part of sialic acid. As the CAM ages, endothelial anionic sites become reduced. That the expression of endothelial anionic domains remained constant despite changes in CAM microvascular permeability in early development (Rizzo et al., 1995a) serves to suggest a minimal role for anionic domains in the development of microvascular permselectivity during normal angiogenesis.