Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

Abstract

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of (125)I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol. min(-1). 10(6) cells(-1). Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-beta-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased (125)I-albumin uptake 2.3-fold. At 37 degrees C, (125)I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC(50) = 1 microM). Using a two-site model, we estimated by Scatchard analysis the affinity (K(D)) and maximal capacity (B(max)) of albumin uptake to be 0.87 microM (K(D1)) and 0.47 pmol/10(6) cells (B(max1)) and 93.3 microM (K(D2)) and 20.2 pmol/10(6) cells (B(max2)). At 4 degrees C, we also observed two populations of specific binding sites, with high (K(D1) = 13.5 nM, 1% of the total) and low (K(D2) = 1.6 microM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.

p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity

“…Our data show an association of p38 MAPK with caveolin-1 in endothelial cells following albumin supplementation of serum-deprived cells, a condition that activates caveolae-mediated endocytosis (11,17). This finding raises the possibility that caveolin-1 is involved in p38 MAPK activation during endocytosis.…”

“…TGF-␤ signaling is triggered by its binding to a heteromeric complex consisting of two transmembrane serine/threonine kinase receptors, type I and type II (T␤RI and T␤RII) (8,16,26). In serum-deprived RLMVEC, we observed that supplementation with albumin induced activation of caveolaemediated endocytosis (11) and subsequent T␤RII signaling that resulted in Smad2 phosphorylation and Smad4 translocation (21,24). These studies did not examine the downstream consequences of activation of endocytosis in promoting endothelial cell proliferation and survival.…”

“…Caveolae internalization is responsible for endocytosis induced by albumin (11). Therefore, to determine the mechanism of p38 MAPK activation, we investigated whether p38 MAPK was associated with caveolin-1, the protein constituent of caveolae.…”

“…THE BASIC FUNCTION of endothelial cells lining the vessel wall is their ability to form a continuous confluent monolayer and serve as a semipermeable barrier (11). Endothelial cells also have a large number of vesicles comprising 15-20% of cell volume (20).…”

Nanoparticle accumulation and transcytosis in brain endothelial cell layers

“…The Au nanoparticles were conjugated with human serum albumin or transferrin to allow potential biological recognition, which, as discussed previously, may affect transcytosis 16,24,63,64 and nanoparticle behavior in general. (BBB cells were also treated with simple PEGylated Au for comparison; some images are shown in Supplementary Fig.…”