Abstract
IntroductionWe previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.MethodsAssay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.ResultsTotal α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.ConclusionsThese data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.
Highlights
We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis
Blocking fut1 expression in rheumatoid arthritis (RA) synovial fibroblasts reduces expression of proangiogenic mediators We found that mRNA expression of monocyte chemoattractant protein 1 (MCP-1)/CCL2, epithelial-derived neutrophil-activating peptide 78 (ENA-78)/CXCL5 and vascular endothelial growth factor (VEGF) in tumor necrosis factor-α (TNF-α)-stimulated fut1 small interfering RNA (siRNA)-transfected RA synovial fibroblasts was significantly decreased compared to control siRNA-transfected RA synovial fibroblasts (Figure 4A, B and C)
We found that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on TNF-α-stimulated fut1 siRNA-transfected RA synovial fibroblasts were decreased compared to control siRNAtransfected or nontreated RA synovial fibroblasts (Figure 5B and C)
Summary
Patients RA and osteoarthritis (OA) ST were obtained from patients undergoing arthroplasty or synovectomy. Total fucosylated proteins from synovial fibroblast conditioned medium and cell lysates were measured by ELLA using 2′FL-BSA as a standard curve. To determine if α(1,2)-linked proteins were expressed on RA ST synovial fibroblasts, mouse anti-human collagen-1 (Abcam, Cambridge, MA, USA) and goat anti-UEA-1 (Vector laboratories Inc.) were used. In vitro cell adhesion assay Adhesion of THP-1 cells to nontreated, control siRNA or fut siRNA treated RA synovial fibroblasts grown to confluence in 96-well plates was examined [25]. Cell surface ELISA for adhesion molecule expression Nontreated, control siRNA-transfected, or fut siRNAtransfected RA synovial fibroblasts (1 × 105/well) were seeded in 96-well plates. Cell proliferation assay Control or fut siRNA-transfected RA synovial fibroblasts were seeded in 96-well plates at 5 × 104 cells/ml. We found that RA ST homogenates contained significantly more fucosylated proteins than did either OA or NL STs (Figure 1A)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
