Kazuko Ogawa

α2,6-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 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.

β-Galactoside α2,6-Sialyltransferase I Cleavage by BACE1 Enhances the Sialylation of Soluble Glycoproteins A NOVEL REGULATORY MECHANISM FOR α2,6-SIALYLATION

BACE1 (β-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid β-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves β-galactoside α2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of α2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of α2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.

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 β 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 β (Aβ) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid β precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aβ. 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.

Molecular insights into β-galactoside α2,6-sialyltransferase secretion in vivo

Beta-galactoside alpha2,6-sialyltransferase (ST6Gal I), which is highly expressed in the liver, is mainly cleaved by Alzheimers beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and secreted into the serum. During our studies to elucidate the molecular mechanism underlying the cleavage and secretion of ST6Gal I, we hypothesized that plasma ST6Gal I may represent a sensitive biomarker for hepatopathological situations. In the present study, we used recently developed sandwich ELISA systems that specifically detect the soluble cleaved form of ST6Gal I in plasma. We found that the level of plasma ST6Gal I was increased in two different types of liver injury models. In zone 1 hepatocyte-injured rats, the level of plasma ST6Gal I was increased together with acute phase reactions. Meanwhile, in zone 3 hepatocyte-injured rats, ST6Gal I secretion was most likely triggered by oxidative stress. Taken together, we propose two possible mechanisms for the upregulation of plasma ST6Gal I in hepatopathological situations: one accompanied by acute phase reactions to increase hepatic ST6Gal I expression and the other triggered by oxidative stress in the liver. We also found that the serum level of ST6Gal I in hepatitis C patients was correlated with the activity of hepatic inflammation.

Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role

Background: N-Glycan α2,6-sialylation regulates the cell surface residency of an anti-apoptotic molecule, platelet endothelial cell adhesion molecule (PECAM). Results: An α2,6-sialylated oligosaccharide inhibited the homophilic PECAM interaction and a cluster-type α2,6-sialyl N-glycan probe bound to PECAM-immobilized beads. Conclusion: PECAM is a weak sialic acid binding lectin. Significance: There is a possibility of using a glycan-based method to modulate angiogenesis. 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.

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.

Development of sandwich enzyme-linked immunosorbent assay systems for plasma β-galactoside α2,6-sialyltransferase, a possible hepatic disease biomarker

Previous reports, including our work, have shown that plasma beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) activity is significantly increased in particular hepatopathological situations, suggesting that it may represent a sensitive biomarker for diagnosing hepatic diseases. So far, activity of ST6Gal I have been measured by using radioactive tracer method in place of measuring amount of ST6Gal I. However, this method is tangled and cannot exclude other sialyltransferase activities. Thus, simple and specific methods for measuring plasma ST6Gal I had been unavailable. Here, we developed two kinds of sandwich enzyme-linked immunosorbent assay (ELISA) systems that specifically detect the soluble cleaved form of ST6Gal I in plasma. In one sandwich ELISA, we detected rat specific sequence, EFQMPK, which is N-terminus of soluble ST6Gal I. In the other sandwich ELISA, we detected internal common sequence among rat, mouse and human ST6Gal I in plasma (M2 ELISA). Using the M2 ELISA, we observed that elevation of plasma ST6Gal I was much faster than elevation of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a carbon tetrachloride (CCl(4))-induced mouse liver injury model. Our data suggest that these ELISA systems are very useful tools for measuring plasma ST6Gal I, which represents a potential biomarker for diagnosing hepatic diseases.

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

Most Alzheimer disease patients show deposition of amyloid β (Aβ) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid β precursor protein (APP) 770, a different APP isoform from neuronal APP695, and that they produce amyloid β peptide. We analyzed the glycosylation of APP770 and found that O‐glycosylated sAPP770 is preferentially processed by proteases for Aβ 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.

Glycosylation controls cooperative PECAM-VEGFR2-β3 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 β3 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-β3, indicating that the reduction of cell surface integrin-β3 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.

Post‐engraftment blood transfusion and outcomes of bone marrow transplantation

Red blood cell (RBC) transfusion prior to allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is linked to poor outcomes, but outcomes related to post‐transplant, especially postengraftment transfusions, have not been fully investigated. We investigated allo‐HSCT outcome in association with early postengraftment transfusion.