Takeo Isozaki

Evidence that CXCL16 is a potent mediator of angiogenesis and is involved in endothelial progenitor cell chemotaxis: Studies in mice with K/BxN serum-induced arthritis

OBJECTIVE To examine the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium. METHODS We utilized the RA synovial tissue SCID mouse chimera system to examine human microvascular EC (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium that was injected intragraft with CXCL16-immunodepleted RA synovial fluid (SF). CXCR6-deficient and wild-type (WT) C57BL/6 mice were primed to develop K/BxN serum-induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression, by immunofluorescence and assessment of CXCL16 signaling activity. RESULTS CXCR6 was prominently expressed on human EPCs and HMVECs, and its expression on HMVECs could be up-regulated by interleukin-1β. SCID mice injected with CXCL16-depleted RA SF exhibited a significant reduction in EPC recruitment. In experiments using the K/BxN serum-induced inflammatory arthritis model, CXCR6(-/-) mice showed profound reductions in hemoglobin levels, which correlated with reductions in monocyte and T cell recruitment to arthritic joint tissue compared to that observed in WT mice. Additionally, HMVECs and EPCs responded to CXCL16 stimulation, but exhibited unique signal transduction pathways and homing properties. CONCLUSION These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand/receptor pair that is closely associated with EPC recruitment and blood vessel formation in the RA joint.

Citrullination of epithelial neutrophil-activating peptide 78/CXCL5 results in conversion from a non-monocyte-recruiting chemokine to a monocyte-recruiting chemokine.

To examine whether the citrullinated chemokines epithelial neutrophil–activating peptide 78 (ENA‐78)/CXCL5, macrophage inflammatory protein 1α/CCL3, and monocyte chemotactic protein 1/CCL2 are detected in the biologic fluid of patients with rheumatoid arthritis (RA), and if so, to determine the biologic activities of these chemokines.

ADAM‐10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis

OBJECTIVE To examine the expression of ADAM-10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. METHODS ADAM-10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM-10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM-10 small interfering RNA (siRNA). To determine whether ADAM-10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. RESULTS Endothelial cells and lining cells within RA ST expressed high levels of ADAM-10 compared with cells within osteoarthritis ST and normal ST. ADAM-10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM-10 siRNA-treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA-treated HMVECs. In addition, ADAM-10 siRNA-treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA-treated HMVECs. CONCLUSION These data show that ADAM-10 is overexpressed in RA and suggest that ADAM-10 may play a role in RA angiogenesis. ADAM-10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

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.

A key role for Fut1-regulated angiogenesis and ICAM-1 expression in K/BxN arthritis

Objectives Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. Methods We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1−/−), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1−/− and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1−/− and wt ECs for adhesion molecule expression. Results Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1−/− ECs formed significantly fewer tubes on Matrigel. Fut1−/− mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1−/− mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1−/− ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1−/− arthritic ankle cryosections compared with wt ankles. Conclusions Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.

Inhibitor of DNA binding 1 as a secreted angiogenic transcription factor in rheumatoid arthritis

IntroductionRheumatoid arthritis (RA) is characterized by enhanced blood vessel development in joint synovium. This involves the recruitment of endothelial progenitor cells (EPCs), allowing for de novo vessel formation and pro-inflammatory cell infiltration. Inhibitor of DNA Binding 1 (Id1) is a transcription factor characteristic of EPCs that influences cell maturation.MethodEnzyme-linked immunosorbant assay (ELISA) and polymerase chain reaction (PCR) were used to examine Id1 levels in synovial fluid (SF) and endothelial cells (ECs), respectively. Immunohistology was used to determine the expression of Id1 in synovial tissue (ST). Human dermal microvascular EC (HMVEC) migration and tube forming assays were used to determine if recombinant human Id1 (rhuId1) and/or RA SF immunodepleted Id1 showed angiogenic activity. We also utilized the RA ST severe combined immunodeficient (SCID) mouse chimera to examine if Id1 recruits EPCs to RA synovium.ResultsST samples immunostained for Id1 showed heightened expression in RA compared to osteoarthritis (OA) and normal (NL) ST. By immunofluorescence staining, we found significantly more Id1 in RA compared to OA and NL vasculature, showing that Id1 expressing cells, and therefore EPCs, are most active in vascular remodeling in the RA synovium. We also detected significantly more Id1 in RA compared to OA and other arthritis SFs by ELISA, which correlates highly with Chemokine (C-X-C motif) ligand 16 (CXCL16) levels. In vitro chemotaxis assays showed that Id1 is highly chemotactic for HMVECs and can be attenuated by inhibition of Nuclear Factor κB and phosphoinositide 3-kinase. Using in vitro Matrigel assays, we found that HMVECs form tubes in response to rhuId1 and that Id1 immunodepleted from RA SF profoundly decreases tube formation in Matrigel in vitro. PCR showed that Id1 mRNA could be up-regulated in EPCs compared to HMVECs in response to CXCL16. Finally, using the K/BxN serum induced arthritis model, we found that EC CXCR6 correlated with Id1 expression by immunohistochemistry.ConclusionsWe conclude that Id1 correlates highly with CXCL16 expression, EPC recruitment, and blood vessel formation in the RA joint, and that Id1 is potently angiogenic and can be up-regulated in EPCs by CXCL16.

Fucosyltransferase 1 Mediates Angiogenesis in Rheumatoid Arthritis

To determine the role of α(1,2)‐linked fucosylation of proteins by fucosyltransferase 1 (FUT1) in rheumatoid arthritis (RA) angiogenesis.

A novel role for inducible Fut2 in angiogenesis

RationaleAngiogenesis plays an important role in wound healing and tumor growth. Fucosyltransferases synthesize fucosylated glycans and may play a major role in vascular biology.ObjectiveTo examine the role of an alpha(1,2) fucosyltransferase (Fut2) in angiogenesis.Methods and resultsWe found that Fut2 mRNA and protein expression is inducible in human dermal microvascular endothelial cells (HMVECs). After finding that Fut2 is inducible in HMVECs, we examined if Fut2 contributes to angiogenesis. We found that Fut2 null endothelial cell (EC) migration and tube formation were significantly less compared to wild type (wt) ECs. Angiogenesis was impaired in Fut2 null compared to wt mice in the mouse Matrigel plug and the sponge granuloma angiogenesis assays. To assess the characteristics of Fut2 null ECs in vivo, we performed Matrigel plug angiogenesis assays in wt mice using Fut2 null and wt mouse ECs. We found a significant decrease in Fut2 null EC incorporation in neoangiogenesis compared to wt ECs. ERK1/2 activation, fibroblast growth factor receptor2, and vascular endothelial growth factor expression were less in Fut2 null ECs, suggesting a possible mechanism of impaired angiogenesis when Fut2 is lacking.ConclusionsThese data suggest a novel role for Fut2 as a regulator of angiogenesis.

Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis

BackgroundInhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified.MethodsWe analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition.ResultsBy PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-β). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo.ConclusionsId1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.

A unique role for galectin-9 in angiogenesis and inflammatory arthritis

BackgroundGalectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis.MethodsTo determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees.ResultsGal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9.ConclusionsGal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.