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.

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TitleRhoA 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.
Publication TypeJournal Article
Year of Publication2008
AuthorsGarnacho, C, Shuvaev, V, Thomas, A, McKenna, L, Sun, J, Koval, M, Albelda, S, Muzykantov, V, Muro, S
JournalBlood
Volume111
Issue6
Pagination3024-33
Date Published2008 Mar 15
ISSN0006-4971
KeywordsActins, Animals, Antibodies, Antigens, CD31, Cells, Cultured, Cytoplasm, Endocytosis, Endothelial Cells, Enzyme Activation, Humans, Male, Mice, Mice, Inbred C57BL, Phosphorylation, rhoA GTP-Binding Protein, Tyrosine
Abstract

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.

DOI10.1182/blood-2007-06-098657
Alternate JournalBlood
PubMed ID18182571
PubMed Central IDPMC2265450
Grant ListHL078785 / HL / NHLBI NIH HHS / United States
HL73940 / HL / NHLBI NIH HHS / United States
P30 DK47757-14 / DK / NIDDK NIH HHS / United States
R01 HL087036-04 / HL / NHLBI NIH HHS / United States
R01 HL087036-05 / HL / NHLBI NIH HHS / United States
R01 HL71175 / HL / NHLBI NIH HHS / United States
R21 HL85533 / HL / NHLBI NIH HHS / United States