CD47-dependent molecular mechanisms of blood outgrowth endothelial cell attachment on cholesterol-modified polyurethane.
We previously showed that blood outgrowth endothelial cells (BOECs) had a high affinity for polyurethane (PU) covalently configured with cholesterol residues (PU-Chol). However, the molecular mechanisms responsible for this enhanced affinity were not determined. CD47, a multifunctional transmembrane glycoprotein involved in cellular attachment, can form a cholesterol-dependent complex with integrin alpha(v)beta(3) and heterotrimeric G proteins. We tested herein the hypothesis that CD47, and the other components of the multi-molecular complex, enhance the attachment of BOECs to PU-Chol. Immunoprecipitation studies, of human and ovine BOECs, demonstrated that CD47 associates with integrin alpha(v) and integrin beta(3) as well as G(alphai-2) protein. The three-fold increase in BOEC attachment to PU-Chol, compared to unmodified PU, was reversed with the addition of blocking antibodies specific for CD47 and integrin alpha(v) and integrin beta(3). Similar results were observed with the addition of methyl-beta-cyclodextrin (MbetaCD), a known disruptor of the CD47 complex as well as of the membrane cholesterol content, to seeded BOEC or PU-Chol films. Reducing CD47 expression, via lentivirus transduced shRNA, decreased BOEC binding to PU-Chol by 50% compared to control groups. These data are the first demonstration of a role for the CD47 cholesterol-dependent signaling complex in BOEC attachment onto synthetic surfaces.
Division of Cardiology, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA.
Masako
M
Ueda
Ivan S
IS
Alferiev
Stacey B
SB
Simons
Robert P
RP
Hebbel
Robert J
RJ
Levy
Stanley J
SJ
Stachelek
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry* / Q000378:metabolism; D051928:CD47 Antigen / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D015536:Down-Regulation; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D011140:Polyurethanes / Q000737:chemistry* / Q000378:metabolism; D012756:Sheep
D001672:Biocompatible Materials; D051928:CD47 Antigen; D039302:Integrin alphaVbeta3; D011140:Polyurethanes; D002784:Cholesterol