RhoA activation and actin reorganization involved in endothelial CAM-mediated endocytosis of anti-PECAM carriers: critical role for tyrosine 686 in the cytoplasmic tail of PECAM-1.
Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a transmembrane glycoprotein involved in leukocyte transmigration, represents a good target for endothelial drug delivery (eg, using antibody-directed nanocarriers, anti-PECAM/NCs). Although endothelial cells do not internalize PECAM antibodies, PECAM-1 engagement by multivalent anti-PECAM conjugates and nanocarriers causes endocytosis via a nonclassic CAM-mediated pathway. We found that endothelial uptake of multivalent anti-PECAM complexes is associated with PECAM-1 phosphorylation. Using model REN cells expressing a series of PECAM-1 deletion and point mutants, we found that the PECAM-1 cytoplasmic domain and, more precisely, PECAM-1 tyrosine 686, is critical in mediating RhoA activation and recruitment of EGFP-RhoA to anti-PECAM/NC binding sites at the plasmalemma, actin polymerization into phalloidin-positive stress fibers, and finally CAM endocytosis of anti-PECAM/NCs. Endothelial targeting and endocytosis of anti-PECAM/NCs were markedly efficient and did not compromise endothelial barrier function in vitro (determined by immunostaining of VE-cadherin and (125)I-albumin transport across endothelial monolayers) or in vivo (determined by electron microscopy imaging of pulmonary capillaries and (125)I-albumin transport from the blood into the lung tissue after intravenous injection of anti-PECAM/NCs in mice). These results reveal PECAM-1 signaling and interactions with the cytoskeleton, which are required for CAM-endocytosis, and may provide safe intra-endothelial drug delivery by anti-PECAM/NCs.
Garnacho
Carmen
C
Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6068, USA.
Shuvaev
Vladimir
V
Thomas
Anu
A
McKenna
Lindsay
L
Sun
Jing
J
Koval
Michael
M
Albelda
Steven
S
Muzykantov
Vladimir
V
Muro
Silvia
S
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D000906:Antibodies; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D014443:Tyrosine; D020742:rhoA GTP-Binding Protein