Caveolae-Dependent and -Independent Uptake of Albumin in Cultured Rodent Pulmonary Endothelial Cells

Abstract

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1-/- mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1-/- mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1-/- MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.

Vesicular uptake of macromolecules by human placental amniotic epithelial cells

“…Fluorophore labeled conjugates were diluted to desired concentrations, centrifuged to remove potential particulates, and vortex mixed prior to treatment. These labeled macromolecules were selected as specific makers: CTB for caveolar vesicles, Tfn for clathrin-coated vesicles, BSA for receptor-mediated vesicular uptake and Dex as a marker for non-specific endocytosis including micro- and macropinocytosis [18, 20, 26, 31–36]. …”

“…Two types of vesicles that may be involved with receptor-mediated fluid transport are caveolar and clathrin-coated vesicles [18–25]. Caveolae are involved in many biological processes including endocytosis and transcytosis and play a major role in transcellular albumin transport [18, 20, 26].…”

“…Caveolae are involved in many biological processes including endocytosis and transcytosis and play a major role in transcellular albumin transport [18, 20, 26]. Clathrin-coated vesicles endocytose fluid, various proteins, essential nutrients and ions, playing a role in cell homeostasis as well as transcellular transport [27–29].…”

“…Our objectives were to use specific fluorophore-labeled macromolecules as markers of receptor mediated and non-receptor mediated vesicular uptake [18, 20, 26, 31–36] to 1) determine whether caveolar and/or clathrin-coated vesicles are present in human amnion cells, 2) analyze the kinetic characteristics of vesicle uptake and 3) determine the spatial distribution of vesicles within amnion cells. Placental amnion cells were used for the study as transport of amniotic fluid and solutes into fetal blood occurs across the placental rather than across the reflected amnion [5, 16].…”

Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

“…It is important to note that inhibitors of the TGF-β type II pathway may have the opposite effect on drug delivery by inhibiting endocytosis of albumin-bound agents. Caveolae are also involved in albumin transcytosis by endothelial cells (47); however, how they respond to TGF-β inhibitors remains unknown, and such mechanisms are beyond the scope of this manuscript.…”