Rie Karasawa

Water-soluble fullerene (C60) inhibits the development of arthritis in the rat model of arthritis.

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the development of inflammation and osteoclastogenesis, suggesting the implication of oxygen free radicals in the pathogenesis of arthritis. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against synovitis in arthritis, both in vitro and in vivo. In the presence or absence of C60 (0.1, 1.0, 10.0 μM), human synovial fibroblasts, synovial infiltrating lymphocytes or macrophages were incubated with tumor necrosis factor-α (TNF-α) (10.0 ng/mL), and the production of proinflammatory cytokines by the individual cells were analyzed. C60 significantly suppressed the TNF-α-induced production of proinflammatory cytokines in synovial fibroblasts, synovial infiltrating lymphocytes and macrophages in vitro. Adjuvant induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 10.0 μM. The left ankle joint was injected intraarticularly with water-soluble C60 (20 μl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 μl), once weekly for eight weeks. Ankle joint tissues were prepared for histological analysis. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced synovitis and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of synovitis and joint destruction with time. These findings indicate that C60 is a potential therapeutic agent for inhibition of arthritis.

Autoantibodies to peroxiredoxin I and IV in patients with systemic autoimmune diseases

Anti‐oxidative enzymes protect living bodies from various oxidative stresses. In the systemic autoimmune diseases, autoantibodies to oxidized molecules and to anti‐oxidative enzymes have been reported. To promote understanding of the relationships between autoimmunity and oxidative stress, we here investigate whether autoimmunity to the anti‐oxidative peroxiredoxin (Prxs) enzymes exists in patients with systemic autoimmune diseases. Specifically, we detected autoantibodies to recombinant Prx I and Prx IV respectively by ELISA and western blotting. Next, clinical parameters were compared between the anti‐Prx I or IV‐positive and ‐negative patients. We found that 33% of the 92 patients with autoimmune diseases tested possessed autoantibodies to Prx I (57% in systemic lupus erythematosus (SLE), 19% in rheumatoid arthritis (RA), 5% in Behçet disease, and 46% in primary vasculitis syndrome). In contrast, autoantibodies to Prx IV were detected in only 17% of the same patients. No significant correlation was found between occurrence of the two autoantibodies. Clinically, possession of anti‐Prx I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Taken together, our data demonstrate the existence of autoantibodies to Prxs for the first time. The autoantibodies to Prx I may be involved in the pathophysiology of systemic autoimmune diseases such as SLE and vasculitis.

Peroxiredoxin 2 is a novel autoantigen for anti‐endothelial cell antibodies in systemic vasculitis

Anti‐endothelial cell antibodies (AECA) have been frequently detected in systemic vasculitis, which affects blood vessels of various sizes. To understand the pathogenic roles of AECA in systemic vasculitis, we attempted to identify target antigens for AECA comprehensively by a proteomic approach. Proteins extracted from human umbilical vein endothelial cells (HUVEC) were separated by two‐dimensional electrophoresis, and Western blotting was subsequently conducted using sera from patients with systemic vasculitis. As a result, 53 autoantigenic protein spots for AECA were detected, nine of which were identified by mass spectrometry. One of the identified proteins was peroxiredoxin 2 (Prx2), an anti‐oxidant enzyme. Frequency of anti‐Prx2 autoantibodies, measured by enzyme‐linked immunosorbent assay (ELISA), was significantly higher in systemic vasculitis (60%) compared to those in collagen diseases without clinical vasculitis (7%, P < 0·01) and healthy individuals (0%, P < 0·01). Further, the titres changed in parallel with the disease activity during time–courses. The presence of anti‐Prx2 autoantibodies correlated significantly with elevation of serum d‐dimers and thrombin–antithrombin complex (P < 0·05). Immunocytochemical analysis revealed that live endothelial cells expressed Prx2 on their surface. Interestingly, stimulation of HUVEC with rabbit anti‐Prx2 antibodies increased secretion of interleukin (IL)‐6, IL‐1β, IL‐1ra, growth regulated oncogene (GRO)‐α, granulocyte colony‐stimulating factor (G‐CSF), granulocyte macrophage colony‐stimulating factor (GM–CSF), IL‐8 and monocyte chemoattractant protein (MCP)‐1 more than twofold compared to that of with rabbit immunoglobulin (Ig)G. Taken together, our data suggest that anti‐Prx2 autoantibodies would be a useful marker for systemic vasculitis and would be involved in the inflammatory processes of systemic vasculitis.

Water-soluble fullerene (C60) inhibits the osteoclast differentiation and bone destruction in arthritis

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the receptor activator NFκB (RANK) signal pathway required for osteoclast differentiation. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against the RANK-induced osteoclastogenesis and osteoclastic bone destruction in arthritis, both in vitro and in vivo. The effects of C60 on the RANK-induced osteoclastogenesis and osteoclastic bone resorption were examined in vitro. Adjuvant-induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 1.0 μM. The left ankle joint was injected intra-articularly with water-soluble C60 (20 μl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 μl) once weekly for eight weeks. Ankle joint tissues were prepared for histologic analysis. C60 significantly inhibited the responses of osteoclast precursor cells to RANK ligand, including osteoclast differentiation and osteoclastic bone resorption in vitro. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced the number of osteoclasts and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of joint destruction with time. These findings indicate that C60 downregulates the RANK-induced osteoclast differentiation and is a potential therapeutic agent for inhibition of osteoclastic bone destruction in arthritis.

Angiogenic growth factors inhibit chondrocyte ageing in osteoarthritis: potential involvement of catabolic stress‐induced overexpression of caveolin‐1 in cellular ageing

Objective:  Recently, attention has been attracted by the finding that overexpression of caveolin‐1 induces cellular senescence in age‐related diseases. We aimed to ascertain whether angiogenic growth factors (AGFs) can inhibit interleukin (IL)‐1β‐induced senescence in human chondrocytes by downregulation of caveolin‐1.

A novel anti‐peroxiredoxin autoantibody in patients with Kawasaki disease

Antibodies to the anti‐oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti‐Prx 2‐positive and ‐negative patients. The presence of anti‐Prx 2 antibodies correlated with a longer period of fever and poor response to high‐dose γ‐globulin therapy in patients with KD. Anti‐Prx 2‐positive patients had significantly greater excretion of urinary 8‐isoprostaglandin than did anti‐Prx 2‐negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.

The DNA Repair Enzyme Apurinic/Apyrimidinic Endonuclease (Apex Nuclease) 2 Has the Potential to Protect against Down-Regulation of Chondrocyte Activity in Osteoarthritis

Apurinic/apyrimidinic endonuclease 2 (Apex 2) plays a critical role in DNA repair caused by oxidative damage in a variety of human somatic cells. We speculated that chondrocyte Apex 2 may protect against the catabolic process of articular cartilage in osteoarthritis (OA). Higher levels of Apex 2 expression were histologically observed in severely compared with mildly degenerated OA cartilage from STR/OrtCrlj mice, an experimental model which spontaneously develops OA. The immunopositivity of Apex 2 was significantly correlated with the degree of cartilage degeneration. Moreover, the OA-related catabolic factor interleukin-1β induced the expression of Apex 2 in chondrocytes, while Apex 2 silencing using small interfering RNA reduced chondrocyte activity in vitro. The expression of Apex 2 in chondrocytes therefore appears to be associated with the degeneration of articular cartilage and could be induced by an OA-related catabolic factor to protect against the catabolic process of articular cartilage. Our findings suggest that Apex 2 may have the potential to prevent the catabolic stress-mediated down-regulation of chondrocyte activity in OA.

Anti-endothelial cell antibodies (AECA) in patients with systemic vasculitis: our research using proteomics

Importance of the field: Anti-endothelial cell antibodies (AECA) may cause damage to endothelial cell (EC) functions and therefore may be of a pathophysiological role in systemic vasculitis. The pathophysiological role of AECA, however, is still uncertain. Areas covered in this review: To clarify the detailed roles of AECA, various methods for identification of target proteins of AECA have been developed, such as expression libraries and proteomic approaches combining two-dimensional electrophoresis and immunoblotting. What the reader will gain: Advances, including our research, have been made in defining the target antigens of AECA, which we summarize in this review. Furthermore, we discuss the possible significance of AECA in the pathophysiology of vascular damage and the value of AECA in systemic vasculitis. Take home message: To identify target antigens of AECA and to establish a standardized method for measuring AECA would be helpful in the search for a possible pathophysiological role of AECA in systemic vasculitis.

Validation of a New Biomarker in Patients with Kawasaki Disease Identified by Proteomics

Systemic vasculitis is a heterogeneous disorder characterized by chronic or acute inflammation, and is classified into groups by the size of the affected blood vessels: large vessel vasculitis, medium vessel vasculitis, or small vessel vasculitis. The pathogenesis of systemic vasculitis is not yet understood and except for Anti-neutrophil Cytoplasmic Antibodies (ANCA) for a subset of small vessel vasculitis, biomarkers for diagnosis and disease activity in systemic vasculitis have still to be discovered. Kawasaki Disease (KD) is an acute systemic vasculitis of infancy and early childhood. It mainly affects small and medium size blood vessels, and can cause cardiovascular complications like coronary artery aneurysm. Anti-endothelial Cell Antibodies (AECA) have been detected in a variety of diseases associated with vascular injury,and KD is one of them. Further, the presence of AECA has been correlated with disease activity, and/or its clinical manifestations [1,2]. A number of mechanisms for AECA, for example, activation and apoptosis of vascular Endothelial Cells (ECs), have been proposed [3]. In KD patients, IgM AECA mediate complement-dependent cytotoxicity against Human Umbilical Vein Endothelial Cells (HUVEC) and gammaglobulin, a standard therapy for KD, may reduce their effect [4]. Grunebaum et al. [5] demonstrated that AECA increased the secretion of Interleukin-6 (IL-6) from HUVEC, in KD patients. However, a significant role for AECA in systemic vasculitis has yet to be found. To evaluate the roles of AECA in detail, it is essential that their target antigens are identified and that the role of individual target antigens is assessed. Various approaches, such as expression libraries and proteomic, have been developed to identify biomarkers [6-11]. In KD, two target antigens for the AECA, tropomyosin and T-plastin, were identified using serological analysis of a recombinant cDNA expression library [11]. However, target antigens suitable for clinical applications, including Myeloperoxidase (MPO) and proteinase 3 of ANCA, remain to be discovered. Proteomics is a powerful tool for detecting and identifying biomarker proteins, because proteomics techniques allow the global analyses of protein function, modifications, composition and dynamics to be performed. In addition, current proteomic techniques can be used to analyze several samples from, for example, serum, urine, cerebrospinal fluid, and various tissues and cells. These kinds of analyses may lead to clinical applications. To identify the target proteins for AECA in patients with vasculitis, we used a proteomics approach that included 2-dimensional electrophoresis and Western Blotting, followed by mass spectrometry [9]. Briefly, we extracted the proteins from HUVEC and HeLa cells (control), and separated them by 2-dimensional electrophoresis to detect AECA antigens specific for ECs. We then analyzed the antigens by Western Blotting against serum samples from patients with vasculitis. We selected the spots that were detected only in the HUVEC samples, and not in HeLa cell samples. This procedure should identify candidate antigens, specific for ECs. We identified the detected proteins by peptide mass fingerprinting. Currently, we have detected more than 150 candidate antigenic spots, and identified more than 50 protein spots. In our earlier study, we identified Peroxiredoxin-2 (Prx2), a member of the Peroxiredoxin (Prx) family of peroxidases, as a target antigen for AECA [9]. In mammalian cells, the Prx family has at least six members (Table 1), and Prx2 is one of the most rapid and potent responders to oxidative stress. Furthermore, oxidative stress has been implicated as a pathogenic factor, and/or a progression-related factor in various diseases, including vasculitis. For these reasons, Prx2 was selected for further investigation. We investigated the clinical significance of anti-Prx2 antibodies in patients with KD. The titers of IgG antibodies to recombinant Prx2 were evaluated by ELISA (Enzymelinked Immunosorbent Assay), using 30 untreated patients with KD, including three patients with Coronary Artery Lesions (CALs), and 15 age- and sex-matched controls. The optical density value of the average ± 2 Standard Deviations (SDs) in the control individuals was defined as 100 AU, and values more than 100 AU, were regarded as positive. IgG antibodies to recombinant Prx2 were detected in 60% (18 of 30) of the untreated patients with KD, whereas no IgG antibodies were detected in the control individuals (Figure 1). We also evaluated the titers of IgM and IgA antibodies to recombinant Prx2 by ELISA, in patients with KD and in controls. IgM antibodies to recombinant Prx2 were

Progress and Clinical Applications in Proteomics

The innovative “omics” technologies such as genomics, transcriptomics, proteomics, and metabolomics have greatly contributed to biomedical discovery and advances. A single gene can engender multiple protein products as a consequence of modulation in the processes of protein production from DNA such as transcription, processing and translation. In addition, protein modifications such as phosphorylation, dephosphorylation, glycosylation, acetylation, sulfation, hydroxylation, carboxymethylation and prenylation occur in vivo. Furthermore, it has been reported that the correlation between mRNA and protein levels was not sufficient to predict protein expression levels from mRNA information