Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM) and are understood to develop under the influence of fibrogenic growth factors. To better understand the detailed mechanisms of persistent fibrosis in SSc, we have previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, transforming growth factor-beta (TGF-beta) transiently induced subcutaneous fibrosis and serial injections of connective tissue growth factor (CTGF) after TGF-beta caused persistent fibrosis. These results suggest that CTGF plays an important role in the development of persistent skin fibrosis and that CTGF may be a potential and specific therapeutic target in skin fibrosis. Therefore, the aim of the current study is to develop a neutralizing monoclonal antibody against human CTGF. We also investigated the neutralizing effect of the antibodies in our animal model. Firstly, by using the DNA immunization method, we developed a panel of anti-CTGF antibodies recognizing the native conformation of human CTGF. Next, to examine the anti-fibrosing effects of these antibodies, newborn B6 mice received subcutaneous injections of TGF-beta for 3 days with either anti-CTGF neutralizing antibodies or control purified immunoglobulin. Anti-CTGF antibodies significantly reduced skin fibrosis and collagen contents compared with the control group. These results suggest that our anti-CTGF antibodies are capable of blocking the development of skin fibrosis at least partially and these anti-CTGF neutralizing antibodies may be useful as the feasible strategy to treat skin fibrotic diseases as SSc.
Since fibrosis is observed in smokers' gingiva, it was hypothesized that fibrosis was caused by nicotine in the periodontium. Therefore, in this study, we investigated the effects of nicotine on the induction of a profibrotic molecule, connective tissue growth factor (CCN2/CTGF), in human gingival fibroblasts (HGFs) and periodontal ligament (PDL) cells. With 1 microg/mL nicotine, vacuolization and attenuated proliferation were observed. Interestingly, 1 microg/mL nicotine increased the production of CCN2/CTGF protein in both cells without increasing mRNA expression. Furthermore, type I collagen mRNA and protein were also increased and were significantly blocked by a CCN2/CTGF neutralizing antibody. This is the first report to describe a relationship between nicotine and CCN2/CTGF in periodontal tissue cells. Analysis of our data also indicated that nicotine was cytotoxic, while it increased CCN2/CTGF and, eventually, type I collagen production. These findings suggest that periodontal fibrosis can be promoted by nicotine from smoking via effects on CCN2/CTGF.
Background: Connective tissue growth factor (CTGF) may be a potential marker of fibrosis. However, platelet-derived CTGF may be released into the plasma by platelet activation during or after blood collection, thereby interfering with accurate determination of the true plasma CTGF level. Plasma CTGF exists as the N-terminal CTGF fragment (N-fragment), composed of modules 1 and 2, whereas platelet CTGF exists as full-length CTGF (full-length), composed of modules 1 -4. We perceived the need to develop a method for distinguishing between the N-fragment and full-length CTGF levels, so that the true plasma and serum CTGF (N-fragment) levels could be accurately determined. Methods: Full-length levels were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies recognizing modules 1 and 4, respectively (M1/4 ELISA). Total CTGF (full-length CTGF plus N-terminal CTGF) levels were determined by a sandwich ELISA using two monoclonal antibodies recognizing modules 1 and 2, respectively (M1/2 ELISA). N-terminal CTGF levels were determined by subtracting the full-length levels from the total CTGF levels. Results: Both the M1/2 and M1/4 ELISAs showed good analytical performance. When the CTGF levels of plasma and serum collected simultaneously from the same subject were compared, the N-fragment levels determined by the subtraction method were the same, in spite of the fact that full-length CTGF was present in the sample. Conclusion: N-fragment levels in plasma and serum can be accurately determined by this subtraction method, even if full-length CTGF in platelets is released during or after blood collection.
Background
Osteoarthritis (OA) is an age-related joint disease characterized by progressive cartilage loss. Synovial mesenchymal stem cells (MSCs) are anticipated as a cell source for OA treatment; however, synovial MSC preparations isolated from OA patients contain many senescent cells that inhibit cartilage regeneration through their senescence-associated secretory phenotype (SASP) and poor chondrogenic capacity. The aim of this study was to improve the biological function of OA synovial MSCs by removing senescent cells using the senolytic drug ABT-263.
Methods
We pretreated synovial MSCs derived from 5 OA patients with ABT-263 for 24 h and then evaluated senescence-associated beta-galactosidase (SA-β-gal) activity, B cell lymphoma 2 (BCL-2) activity, apoptosis, surface antigen expression, colony formation ability, and multipotency.
Results
The ABT-263 pretreatment significantly decreased the percentage of SA-β-gal-positive cells and BCL-2 expression and induced early- and late-stage apoptosis. Cleaved caspase-3 was expressed in SA-β-gal-positive cells. The pretreated MSCs formed greater numbers of colonies with larger diameters. The expression rate of CD34 was decreased in the pretreated cells. Differentiation assays revealed that ABT-263 pretreatment enhanced the adipogenic and chondrogenic capabilities of OA synovial MSCs. In chondrogenesis, the pretreated cells produced greater amounts of glycosaminoglycan and type II collagen and showed lower expression of senescence markers (p16 and p21) and SASP factors (MMP-13 and IL-6) and smaller amounts of type I collagen.
Conclusion
Pretreatment of synovial MSCs from OA patients with ABT-263 can improve the function of the cells by selectively eliminating senescent cells. These findings indicate that ABT-263 could hold promise for the development of effective cell-based OA therapy.
Mesenchymal stem cells (MSCs) can show trisomy 7; however, the safety of these cells has not been fully investigated. The purposes of this study were to determine the ratio of patients whose synovial MSCs were transplanted clinically, to intensively investigate MSCs with trisomy 7 from a safety perspective, and to follow up the patients for 5 years after transplantation. Synovial MSCs at passage 0 were transplanted into a knee for degenerative meniscus tears in 10 patients, and the patients were checked at 5 years. The synovial MSCs were evaluated at passages 0 to 15 by G-bands and digital karyotyping, and trisomy 7 was found in 3 of 10 patients. In those 3 patients, 5% to
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