Abstract
Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that α2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on α2,6-sialic acid. We also found that the absence of α2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.
Highlights
Angiogenesis is a physiological process that encompasses the growth of capillary blood vessels, and the disturbance of angiogenesis contributes to the pathogenesis of numerous disorders, including cancer [1]
platelet endothelial cell adhesion molecule (PECAM) Expression Is Altered by ST6Gal I Deficiency—Based on previous reports that endothelial cells express significant levels of ␣2,6-linked sialic acid, which are increased by inflammatory cytokines [15, 17], we tried to elucidate a role for ␣2,6linked sialic acid in endothelial functions
We found that ST6Gal I itself was exclusively detected in the blood vessels
Summary
Materials—The sources of the materials used in this study were as follows: tissue culture media and reagents, including Dulbecco’s modified Eagle’s medium/F-12, from Invitrogen; FuGENE 6 and recombinant N-glycosidase F from Roche Applied Science; protein A-Sepharose Fast Flow from GE Healthcare; protein molecular weight standards from Bio-Rad; and all other chemicals from Sigma or Wako Chemicals. Brain sections were incubated with 0.3% hydrogen peroxidase in methanol, treated with the blocking solutions supplied in a tyramide signal amplification kit (TSA Biotin System; PerkinElmer Life Sciences), and incubated with anti-PECAM antibodies MEC13.3 (BD Biosciences) or M-20 (Santa Cruz Biotechnology) diluted 1:500 in TBS (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl) overnight at 4 °C. After three rinses with TNT buffer (TBS containing 0.05% Tween 20) for 5 min each, the sections were incubated with biotinylated TJA-I lectin (1:1000 dilution; Honen Co.) plus Alexa Fluor 546-conjugated goat anti-rat IgG (1:100 dilution; Molecular Probes) for 45 min. The cells (5 ϫ 105) were incubated with SSA-fluorescein isothiocyanate (10 g/ml) or an anti-PECAM-PE antibody (5 g/ml; Santa Cruz Biotechnology) for 30 min on ice, washed three times, and suspended in FACS buffer. A luminogenic substrate containing an Asp-Glu-Val-Asp sequence was added and incubated at 37 °C for 1 h, followed by analysis using a multiwell luminometer
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