Keum Hee Sa

Salvia plebeia extract inhibits the inflammatory response in human rheumatoid synovial fibroblasts and a murine model of arthritis

Salvia plebeia R. Br. has been used to treat a variety of inflammatory diseases and as an antioxidant in many countries, including Korea and China. In this study, we investigated the effects of S. plebeia extract (SPE) on inflammatory arthritis and the underlying mechanisms of action. We used a collagen-induced arthritis (CIA) mouse model. TNF-α-stimulated rheumatoid arthritis (RA) synovial fibroblasts were used to elucidate the underlying mechanisms of action. Oral administration of SPE improved the clinical arthritis score, footpad thickness, and histologic changes, as well as serum IgG1 and IgG2a levels. SPE administration inhibited Th1/Th2/Th17 phenotype CD4(+) T lymphocyte expansion in inguinal lymph node and expression of inflammatory mediators such as cytokines, MMP-1, and MMP-3 in the ankle joint tissue. SPE significantly suppressed the expression of cytokines and MMP-1 by down-regulating NF-κB, Akt, and mitogen-activated protein kinases in RA synovial fibroblasts. Taken together, these results indicate that SPE is therapeutically efficacious against chronic inflammatory arthritis, suggesting that SPE is a candidate for treating RA.

A matrix metalloproteinase 1-cleavable composite peptide derived from transforming growth factor β-inducible gene h3 potently inhibits collagen-induced arthritis.

OBJECTIVE Transforming growth factor β-inducible gene h3 (βIG-H3), which is abundantly expressed in rheumatoid synovium, and matrix metalloproteinases (MMP) play important roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the therapeutic efficacy of βIG-H3-derived peptides using MMP-1-dependent target tissue delivery in chronic inflammatory arthritis. METHODS Peptides developed from βIG-H3 derivatives, including the second and fourth YH peptides, the fourth fas-1 domain, the fourth fas-1 domain truncated for H1 and H2 sequences (dhfas-1), and an MMP-1- cleavable composite peptide (MFK24), were cloned. We confirmed the specificity of MFK24 cleavage by immunoblot analysis after treatment with different proteases. RESULTS The YH18 peptide in the fourth fas-1 domain of βIG-H3 was weakly effective in suppressing arthritis severity in mice with collagen-induced arthritis (CIA). Treatment with higher-dose dhfas-1 (30 mg/kg) showed remarkable efficacy, whereas treatment with a lower dose (10 mg/kg) resulted in only partial improvement. MFK24, a composite peptide consisting of dhfas-1 and RGD peptide linked by MMP-1 substrate, was cleaved specifically by MMP-1. The adhesion and migration of NIH3T3 cells mediated by βIG-H3 were inhibited by MFK24 at a low concentration. MFK24 suppressed the adhesion of NIH3T3 cells more efficiently compared with murine dhfas-1 (MFK00) or RGD, either alone or in combination. The therapeutic efficacy of MFK24 in mice with CIA was remarkably enhanced, with consistently reduced expression of inflammatory mediators within joint tissue. CONCLUSION This proof-of-concept study showed that an MMP-cleavable composite peptide, based on βIG-H3 derivatives, had markedly improved therapeutic efficacy in chronic inflammatory arthritis, implicating a new expandable strategy for enhancement of the efficacy of 2 different active molecules in RA.

FCRL3 gene polymorphisms contribute to the radiographic severity rather than susceptibility of rheumatoid arthritis.

The ethnic heterogeneity and genetic complexity of rheumatoid arthritis (RA) have produced inconsistent results in previous genetic association studies concerning FCRL3. This study sought to delineate the association between the FCRL3 gene polymorphisms and susceptibility to RA and to investigate the effects of the polymorphisms on the progression of joint destruction in RA. RA patients (n = 377) and healthy unrelated controls (n = 298) were recruited. Genotyping of -169 T>C and -110 G>A in the promoter and 1,381 G>A in the intron was accomplished using FRET assays. The distribution of genotypes and haplotypes did not differ between RA patients and controls. When we investigated the role of FCRL3 polymorphisms for the severity of RA, patients with the CC genotype in the -169 T>C polymorphism had a higher modified Sharp score than other genotype groups (p = 0.034) among patients with disease duration ≥10 years. The slope of the regression line for modified Sharp score over disease duration (10.12/year) was significantly steeper in patients with the CC genotype than in the T carriers (5.69/year) at the -169 T>C polymorphism (p = 0.003), indicating the faster progression of radiologic destruction in the CC genotype. In conclusion, polymorphisms of the FCRL3 gene may contribute to the progression of joint destruction rather than susceptibility of RA.

Intracellular delivery of desulfated heparin with bile acid conjugation alleviates T cell-mediated inflammatory arthritis via inhibition of RhoA-dependent transcellular diapedesis

Heparin has a potential regulatory role in inflammatory diseases. However, the anticoagulant activity and poor oral bioavailability of heparin limit its use as an anti-inflammatory agent. Conjugation of bis-deoxycholic acid to 6-O-desulfated low molecular weight heparin (6DSHbD) was efficiently internalized by activated endothelial cells via a 2-step model, in which heparin attaches to adhesion molecules that facilitate accessibility of the bile acid conjugate to membrane transporters. The critical role of P-selectin during endothelial cell uptake of 6DSHbD by arthritic tissue was confirmed in p-selectin(-/-) arthritic mice. Intracellular 6DSHbD inhibited transcellular diapedesis of T cells through activated endothelial cells and impaired both the formation of ICAM-1-rich docking structures at the T cell contact surface and subsequent cytoskeletal rearrangement. Furthermore, 6DSHbD blocked activation of RhoA-GTPase and phosphorylation of ezrin/radixin/moesin induced by ICAM-1 cross-linking on activated endothelial cells, thereby impairing lymphocyte transcellular transmigration. After oral administration 6DSHbD was preferentially delivered to inflamed joint tissue, particularly in and around post-capillary venular endothelium and inhibited effector T cell homing to arthritic joints. Aggravation of collagen-induced arthritis conferred by the transfer of effector T cells was suppressed by oral 6DSHbD. Thus, intracellular heparin exerts anti-inflammatory effects through the inhibition of RhoA-dependent transendothelial recruitment of T cells and may have applications in the treatment of chronic inflammatory arthritis.

In vivo quantitative measurement of arthritis activity based on hydrophobically modified glycol chitosan in inflammatory arthritis: more active than passive accumulation.

We demonstrated that arthritis could be visualized noninvasively using hydrophobically modified glycol chitosan nanoparticles labeled with Cy5.5 (HGC-Cy5.5) and an optical imaging system. Activated macrophages expressing Mac-1 molecules effectively phagocytosed HGC-Cy5.5, which formed spherical nanoparticles under physiologic conditions. We estimated the applicability of HGC-Cy5.5 to quantitative analysis of arthritis development and progression. Near-infrared fluorescence images, captured after HGC-Cy5.5 injection in mice with collagen-induced arthritis, showed stronger fluorescence intensity in the active arthritis group than in the nonarthritis group. According to the progression of arthritis in both collagen-induced arthritis and collagen antibody-induced arthritis models, total photon counts (TPCs) increased in parallel with the clinical arthritis index. Quantitative analysis of fluorescence after treatment with methotrexate showed a significant decrease in TPC in a dose-dependent manner. Histologic evaluation confirmed that the mechanism underlying selective accumulation of HGC-Cy5.5 within synovitis tissues included enhanced phagocytosis of the probe by Mac-1-expressing macrophages as well as enhanced permeability through leaky vessels. These results suggest that optical imaging of arthritis using HGC-Cy5.5 can provide an objective measurement of disease activity and, at the same time, therapeutic responses in rheumatoid arthritis.

Stepwise inhibition of T cell recruitment at post-capillary venules by orally active desulfated heparins in inflammatory arthritis

Identification of the structure-function relationship of heparin, particularly between 2-O-, 6-O-, and N-sulfation and its anticoagulant or anti-inflammatory activities, is critical in order to evaluate the biological effects of heparin, especially in conjunction with modifications for oral formulation. In this study, we demonstrated that removal of 2-O, 6-O, or N-desulfation and their hydrophobic modifications have differential effects on the blocking of interactions between sLeX and P-and L-selectins, with highest inhibition by 6-O desulfation, which was consistent with their in vivo therapeutic efficacies on CIA mice. The 6-O desulfation of lower molecular weight heparin (LMWH) retained the ability of LMWH to interfere with T cell adhesion via selectin-sLeX interactions. Furthermore, 6DSHbD coated on the apical surface of inflamed endothelium directly blocked the adhesive interactions of circulating T cells, which was confirmed in vivo by suppressing T cell adhesion at post-capillary venular endothelium. Thus, in series with our previous study demonstrating inhibition of transendothelial migration, oral delivery of low anticoagulant LMWH to venular endothelium of inflamed joint tissues ameliorated arthritis by the stepwise inhibition of T cell recruitment and provides a rationale for the development of modified oral heparins as innovative agents for the treatment of chronic inflammatory arthritis.

Robust Therapeutic Efficacy of Matrix Metalloproteinase-2-Cleavable Fas-1-RGD Peptide Complex in Chronic Inflammatory Arthritis

Objective Therapeutic agents that are transformable via introducing cleavable linkage by locally enriched MMP-2 within inflamed synovium would enhance therapeutic efficacy on chronic inflammatory arthritis. Transforming growth factor-β-inducible gene-h3 (βig-h3), which consists of four fas-1 domains and an Arg-Gly-Asp (RGD) motif, intensifies inflammatory processes by facilitating adhesion and migration of fibroblast-like synoviocyte in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to investigate whether a MMP-2-cleavable peptide complex consisting of a fas-1 domain and an RGD peptide blocks the interaction between βig-h3 and resident cells and leads to the amelioration of inflammatory arthritis. Methods We designed βig-h3-derivatives, including the fourth fas-1 domain truncated for H1 and H2 sequences of mouse (MFK00) and MMP-2-cleavable peptide complex (MFK902). MMP-2 selectivity was examined by treatment with a series of proteases. MFK902 efficacy was determined by the adhesion and migration assay with NIH3T3 cells in vitro and collagen-induced arthritis (CIA) model using male DBA/1J mice in vivo. The mice were treated intraperitoneally with MFK902 at different dosages. Results MFK902 was specifically cleaved by active MMP-2 in a concentration-dependent manner, and βig-h3-mediated adhesion and migration were more effectively inhibited by MFK902, compared with RGD or MFK00 peptides. The arthritis activity of murine CIA, measured by clinical arthritis index and incidence of arthritic paws, was significantly ameliorated after treatment with all dosages of MFK902 (1, 10, and 30 mg/kg). MFK902 ameliorated histopathologic deterioration and reduced the expression of inflammatory mediators simultaneously with improvement of clinical features. In addition, a favorable safety profile of MFK902 was demonstrated in vivo. Conclusion The present study revealed that MMP-2-cleavable peptide complex based on βig-h3 structure is a potent and safe therapeutic agent for chronic inflammatory arthritis, thus providing reliable evidence for a MMP-2-cleavable mechanism as a tissue-targeted strategy for treatment of RA.