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Live dynamic imaging of caveolae pumping targeted antibody rapidly and specically across endothelium in the lung
http://www.nature.com/naturebiotechnology
Phil Oh1, Per Borgstrm1, Halina Witkiewicz1, Yan Li1, Bengt J Borgstrm2, Adrian Chrastina1, Koji Iwata3, Kurt R Zinn4, Richard Baldwin1, Jacqueline E Testa1 & Jan E Schnitzer1
How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy conrms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital uorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body c-scintigraphic imaging shows rapid, specic delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafcking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. In vivo proteomic imaging as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.
Caveolae are caveolin-coated, omega-shaped plasmalemmal invaginations 6070 nm in diameter that bud from the plasma membrane in a dynamin and GTP-dependent manner1,2. They are especially abundant in vascular endothelia, where they function in endocytosis and transcytosis to trafc select macromolecules and to maintain tissue homeostasis. Caveolin knockout mice exhibit poor endothelial cell barrier function with compensatory tissue disruption and edema, particularly evident in the lung3,4. The study of trafcking
by caveolae has been hampered by a lack of caveolae-specic probes.
This is especially true for the caveolae of endothelial cells, which in cell culture exhibit phenotypic drift, including altered protein expression5,6 and a greater than tenfold decrease in caveolae density7.Studies of caveolae trafcking in many types of cultured cells have suggested that caveolae mediate endocytosis at a much slower rate than that observed for clathrin-mediated trafcking (12 h versus 510 min)810. Caveolae have even been described as static structures that do not constitutively trafc cargo1113. In vivo data on caveolae
trafcking are conspicuously lacking. Electron microscopy (EM)...