Glycosylation controls cooperative PECAM-VEGFR2-ß3 integrin functions at the endothelial surface for tumor angiogenesis.

Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1-/- mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1-/- endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin ß3 was disturbed in St6gal1-/- mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-ß3, indicating that the reduction of cell surface integrin-ß3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.

In Situ Ligation of High- and Low-Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition.

This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high- and low-affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low-affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high-affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets.

Cell surface and in vivo interaction of dendrimeric N-glycoclusters.

While many examples have been reported that glycoclusters interact with target lectins more strongly than single molecules of glycans, through multivalency effects, literature examples to support lectin interactions/modulations on cell surface and in live animals is quite rare. Our N-glycoclusters, which were efficiently prepared by immobilizing 16 molecules of the asparagine-linked glycans (N-glycans) onto a lysine-based dendron template through histidine-mediated Huisgen cycloaddition, were shown to efficiently detect platelet endothelial cell adhesion molecule (PECAM) on human umbilical vein endothelial cells (HUVEC) as a α(2-6)-sialylated oligosaccharides recognizing lectin. Furthermore, the identity of the N-glycans on our N-glycoclusters allowed control over organ-selective accumulation and serum clearance properties when intravenously injected into mice.

Sweet role of platelet endothelial cell adhesion molecule in understanding angiogenesis.

The vascular endothelial glycocalyx contains several anionic sugars, one of which is a sialic acid attached to both N- and O-glycans. Platelet endothelial cell adhesion molecule (PECAM), a member of the Ig superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis and angiogenesis, has recently been shown to recognize α2,6-sialic acid. In endothelial cells that lack α2,6-sialic acid because of sialyltransferase ST6Gal I deficiency, impairment of the homophilic PECAM interaction and PECAM-dependent cell survival signaling is observed. In this review, we will introduce part of the biological role of PECAM, and discuss how the lectin activity of PECAM is related to angiogenesis.

Interaction of platelet endothelial cell adhesion molecule (PECAM) with α2,6-sialylated glycan regulates its cell surface residency and anti-apoptotic role.

The luminal sides of vascular endothelial cells are heavily covered with a so-called glycocalyx, but the precise role of the endothelial glycocalyx remains unclear. Our previous study showed that N-glycan α2,6-sialylation regulates the cell surface residency of an anti-apoptotic molecule, platelet endothelial cell adhesion molecule (PECAM), as well as the sensitivity of endothelial cells toward apoptotic stimuli. As PECAM itself was shown to be modified with biantennary N-glycans having α2,6-sialic acid, we expected that PECAM would possess lectin-like activity toward α2,6-sialic acid to ensure its homophilic interaction. To verify this, a series of oligosaccharides were initially added to observe their inhibitory effects on the homophilic PECAM interaction in vitro. We found that a longer α2,6-sialylated oligosaccharide exhibited strong inhibitory activity. Furthermore, we found that a cluster-type α2,6-sialyl N-glycan probe specifically bound to PECAM-immobilized beads. Moreover, the addition of the α2,6-sialylated oligosaccharide to endothelial cells enhanced the internalization of PECAM as well as the sensitivity to apoptotic stimuli. Collectively, these findings suggest that PECAM is a sialic acid binding lectin and that this binding property supports endothelial cell survival. Notably, our findings that α2,6-sialylated glycans influenced the susceptibility to endothelial cell apoptosis shed light on the possibility of using a glycan-based method to modulate angiogenesis.

Polyamine modification by acrolein exclusively produces 1,5-diazacyclooctanes: a previously unrecognized mechanism for acrolein-mediated oxidative stress.

Acrolein, a toxic unsaturated aldehyde generated as a result of oxidative stress, readily reacts with a variety of nucleophilic biomolecules. Polyamines, which produced acrolein in the presence of amine oxidase, were then found to react with acrolein to produce 1,5-diazacyclooctane, a previously unrecognized but significant downstream product of oxidative stress. Although diazacyclooctane formation effectively neutralized acrolein toxicity, the diazacyclooctane hydrogel produced through a sequential diazacyclooctane polymerization reaction was highly cytotoxic. This study suggests that diazacyclooctane formation is involved in the mechanism underlying acrolein-mediated oxidative stress.

A cascading reaction sequence involving ligand-directed azaelectrocyclization and autooxidation-induced fluorescence recovery enables visualization of target proteins on the surfaces of live cells.

A general probe designed to induce a cascading sequence of reactions on a target protein was efficiently synthesized. The cascading reaction sequence involved (i) ligand-directed azaelectrocyclization with lysine and (ii) the autooxidation-induced release of a fluorescence quencher from the labeled protein. The probe was linked to a cyclic RGDyK peptide to enable the selective visualization of integrin αVß3 on the surfaces of live cells.

Soluble amyloid precursor protein 770 is a novel biomarker candidate for acute coronary syndrome.

Most Alzheimer disease patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and that they produce amyloid ß peptide. We analyzed the glycosylation of APP770 and found that O-glycosylated sAPP770 is preferentially processed by proteases for Aß production. Because the soluble APP cleavage product sAPP is considered to be a possible marker for Alzheimer disease diagnosis, sAPP, consisting of a mixture of these variants, has been widely measured. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable us to discriminate between endothelial and neurological dysfunctions. Our recent findings, showing that the level of plasma sAPP770 is significantly higher in patients with acute coronary syndrome, raise the possibility that sAPP770 could be an indicator of endothelial dysfunction. In this review, we first describe the expression, glycosylation, and processing of APP770, and then discuss sAPP770 as a novel biomarker candidate of acute coronary syndrome.

Soluble amyloid precursor protein 770 is released from inflamed endothelial cells and activated platelets: a novel biomarker for acute coronary syndrome.

BACKGROUND: Separate monitoring of the cleavage products of different amyloid ß precursor protein (APP) variants may provide useful information. RESULTS: We found that soluble APP770 (sAPP770) is released from inflamed endothelial cells and activated platelets as judged by ELISA. CONCLUSION: sAPP770 is an indicator for endothelial and platelet dysfunctions. SIGNIFICANCE: How sAPP770 is released in vivo has been shown. Most Alzheimer disease (AD) patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aß. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.

A unique N-glycan on human transferrin in CSF: a possible biomarker for iNPH.

Idiopathic normal pressure hydrocephalus (iNPH) is an elderly dementia caused by abnormal metabolism in the cerebrospinal fluid (CSF). The tap test is the current basis for confirming iNPH, but it shows very low sensitivity, indicating that many patients who might be cured by a shunt operation will be missed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found two transferrin isoforms: one had a unique N-glycan (Tf-1) whereas the other had N-glycan similar to that of serum transferrin (Tf-2). Glycan analyses revealed that Tf-1 had branching (biantennary) asialo- and agalacto-complex type N-glycans (N-acetylglucosamine [GlcNAc]-terminated glycans), which carried bisecting ß1,4-N-acetylglucosamine and core α1,6-fucose. To examine glycoform whether changes in iNPH, we introduced the Tf-2/Tf-1 ratio as a diagnostic index, which minimized blot-to-blot variations in measurement. The Tf-2/Tf-1 ratios of iNPH patients are significantly higher than those of controls (p = 0.0019) and Alzheimer's patients (p = 0.0010). This suggests that the Tf-2/Tf-1 ratio could distinguish iNPH from Alzheimer's disease, and possibly other dementias. In conclusion, glycoform analysis has diagnostic potential in neurological diseases.

Alpha2,6-sialic acid on platelet endothelial cell adhesion molecule (PECAM) regulates its homophilic interactions and downstream antiapoptotic signaling.

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 alpha2,6-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 alpha2,6-sialic acid. We also found that the absence of alpha2,6-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.