THU0019 INHIBITION OF FUCOSYLATION IN ENDOTHELIAL CELLS REDUCES RHEUMATOID ARTHRITIS ANGIOGENESIS
Authors: Takeo Isozaki, Shinichiro Nishimi, Tsuyoshi Kasama
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Glycosylation has been reported to associate with tumor invasion and metastasis. Fucosylation is involved the biological functions of adhesion molecules and growth factor receptors. In regards to arthritis, we have previously reported that fucosylated proteins were expressed in rheumatoid arthritis (RA) synovial tissues. However, a direct role for fucosylated cytokines in RA has not been demonstrated. <h3>Objectives:</h3> To confirm that fucosylated proteins are expressed in RA and are involved in RA angiogenesis. <h3>Methods:</h3> Total glycans were determined in serum from normal (NL) subjects and RA patients using mass spectrometry. N-glycans from 10 RA or 10 healthy controls, and 10 pretreatment with tocilizumab (TCZ) or after treatment sera at 24 weeks were purified by glycoblotting using BlotGlycoH.To determine whether fcosylated proteins involved with RA inflammation, the correlation with disease activity score (DAS) 28 (ESR) was measured. 2-deoxy-D-galactose (2-dGal) is an analog of hexose that inhibits fucosylation. In order to confirm the role of fucosylation in RA angiogenesis, we did Matrigel assays <i>in vitro</i>. To block the expression of fcosylated proteins, human umbilical vein endothelial cells (HUVECs) were treated with 2-dGal (15 mM) for 5 days. After treatment with 2-dGal, HUVECs were plated on Matrigel and were incubated with phosphate buffered saline (PBS) or RA synovial fluids. In addition, chemotaxis assays were performed to determine the role of fucosylation in HUVEC migration. Finally, expression of proangiogenic cytokines such as fractalkine/CX3CL1, CXCL16, interleukin (IL)-8/CXCL8, monocyte chemotactic protein 1 (MCP-1)/CCL2, epithelial neutrophil-activating protein 78 (ENA-78)/CXCL5 and vascular endothelial growth factor (VEGF) in 2-dGal treated HUVEC conditioned medium were measured by ELISA. <h3>Results:</h3> Total glycans in RA serum were significantly higher than in NL serum [mean ± SEM; 477 ± 24 pmol/μl (n=10) and 339 ± 14 pmol/μl (n=10), p&lt;0.05, respectively]. In addition, total glycans in RA serum were significantly decreased with tocilizumab treatment at 24 weeks. Total glycans in RA serum were also correlated with DAS28 (ESR). Percent of fucosylated proteins in total glycans were decreased with TCZ treatment at 24 weeks. 2-d Gal treated HUVEC tube formed towards RA synovial fluids (n=6 patients) were decreased compared with nontreated HUVEC tube formed (number of tube formed; 7 ± 1 and 25 ± 2, p&lt;0.05, respectively). We found that 2dGal treated HUVECs showed decreased migration compared with nontreated HUVECs, towards RA SF (numbers of HUVEC migrated; 3 ± 1 and 34 ± 4, n=5 patients, p&lt;0.05). Fractalkine/CX3CL1, CXCL16, or IL-8/CXCL8 in 2-d Gal treated HUVEC conditioned medium were decreased compared with in non-treated HUVEC conditioned medium but not MCP-1/CCL2, ENA-78/CXCL5, or VEGF. <h3>Conclusion:</h3> These data indicate that glycoproteins are involved with RA, and play a role in angiogenesis in RA and suggest that targeting glycosylation especially fucosylation may provide a method by which to decrease inflammation and potentially treat other inflammatory diseases. <h3>Disclosure of Interests:</h3> None declared