Harumoto Yamada

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [3H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation withp-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.

Macrophage- and neutrophil-dominant arthritis in human IL-1α transgenic mice

To study the effects of IL-1α in arthritis, we generated human IL-1α (hIL-1α). Transgenic mice expressed hIL-1α mRNA in various organs, had high serum levels of hIL-1α, and developed a severe polyarthritic phenotype at 4 weeks of age. Not only bone marrow cells but also synoviocytes from knee joints produced biologically active hIL-1α. Synovitis started 2 weeks after birth, and 8-week-old mice showed hyperplasia of the synovial lining layer, the formation of hyperplastic synovium (pannus) and, ultimately, destruction of cartilage. Hyperplasia of the synovial lining was due to the accumulation of macrophage-like cells expressing F4/80 molecules. hIL-1α was widely distributed in macrophage- and fibroblast-like cells of the synovial lining cells, as well as synovial fluid monocytes. T and B cells were rare in the synovial fluid, and analysis of marker expression suggests that synoviocytes were directly histolytic and did not act as antigen-presenting cells. In the joints of these mice, we found elevated levels of cells of the monocyte/macrophage and granulocyte lineages and of polymorphonuclear neutrophils (PMNs), most of which expressed Gr-1, indicating that they were mature, tissue-degrading PMNs. Cultured synoviocytes and PMNs from these animals overexpress GM-CSF, suggesting that the hematopoietic changes induced by IL-1 and the consequent PMN activation and joint destruction are mediated by this cytokine.

Membrane-Associated IL-1 Contributes to Chronic Synovitis and Cartilage Destruction in Human IL-1α Transgenic Mice

IL-1 molecules are encoded by two distinct genes, IL-1α and IL-1β. Both isoforms possess essentially identical activities and potencies, whereas IL-1α, in contrast to IL-1β, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1α (hIL-1α) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys64 to Ala271 of the hIL-1α amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1α. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1α activity or concentration of serum hIL-1α. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1α, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.

Identification and characterization of PDGFRα+ mesenchymal progenitors in human skeletal muscle.

Fatty and fibrous connective tissue formation is a hallmark of diseased skeletal muscle and deteriorates muscle function. We previously identified non-myogenic mesenchymal progenitors that contribute to adipogenesis and fibrogenesis in mouse skeletal muscle. In this study, we report the identification and characterization of a human counterpart to these progenitors. By using PDGFRα as a specific marker, mesenchymal progenitors can be identified in the interstitium and isolated from human skeletal muscle. PDGFRα+ cells represent a cell population distinct from CD56+ myogenic cells, and adipogenic and fibrogenic potentials were highly enriched in the PDGFRα+ population. Activation of PDGFRα stimulates proliferation of PDGFRα+ cells through PI3K-Akt and MEK2-MAPK signaling pathways, and aberrant accumulation of PDGFRα+ cells was conspicuous in muscles of patients with both genetic and non-genetic muscle diseases. Our results revealed the pathological relevance of PDGFRα+ mesenchymal progenitors to human muscle diseases and provide a basis for developing therapeutic strategy to treat muscle diseases.

Cementless total hip replacement: past, present, and future

Cementless total hip replacement (THR) is rapidly being accepted as the surgery for arthritic diseases of the hip joint. The bone-ingrowth rate in porous-type cementless implants was about 90% over 10 years after surgery, showing that biological fixation of cementless THR was well maintained on both the stem and cup sides. As for the stress shielding of the femur operated using a distal fixation-type stem, severe bone resorption was observed. The severe bone resorption group showed continuous progression for more than 10 years after surgery. Stem loosening directly caused by stress shielding has been considered less likely; however, close attention should be paid to bone resorption-associated disorders including femoral fracture. Cementless cups have several specific problems. It is difficult to decide whether a cup should be placed in the physiological position for the case of acetabular dysplasia by bone grafting or at a relatively higher position without bone grafting. The bone-ingrowth rate was lower in the group with en bloc bone grafting, and the reactive line was frequently noted in the bone-grafted region. Although no data indicated that en bloc bone grafting directly led to poor outcomes, such as loosening, cup placement at a higher site without bone grafting is now selected by most operators. The polyethylene liner in a cementless cup is thinned due to the metal cup thickness; however, it has been suggested that the apparent relation between the cup size and the wear rate was absent as long as a cementless cup is used. Comparative study indicated cementless THR was inferior with regard to the yearly polyethylene wear rate and incidence of osteolysis on both the stem and cup sides. Meta-analysis study on the survival rate between cement and cementless THR reported that cemented THR was slightly superior. It should be considered that specific problems for cementless THR, especially with regard to polyethylene wear, do occur.

Matrix metalloproteinase-3 production by human degenerated intervertebral disc

To assess its possible role in the pathophysiology of intervertebral disc degeneration, we investigated the production of matrix metalloproteinase-3 (MMP-3) using human intervertebral disc explant culture. Five normal and 10 degenerated disc specimens were used. The levels of MMP-3 released in the medium were measured with use of an enzyme immunoassay. The results showed that the level of MMP-3 in the degenerated group (0.57 microg/ml/mg wet weight; n = 10) was significantly higher than that of the control group (0.29 microg/ml/mg wet weight; n = 5) (p < 0.05). Immunostaining of MMP-3 revealed that the ratio of positive staining cells in the degenerated group was greater than that of the control group. These observations suggest that MMP-3 produced by human intervertebral disc may be involved in the intervertebral disc degeneration, particularly in the initiation of matrix degradation of intervertebral disc.

Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56+ and PDGFRα+ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56+ cells and PDGFRα+ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα+ cells formed bone-like tissue and showed successful engraftment, while CD56+ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα+ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα+ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα+ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα+ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα+ cells. Our results suggest that PDGFRα+ cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα+ cells.

Multiinstitutional epidemiological study regarding osteoarthritis of the hip in Japan

Background. Osteoarthritis (OA) of the hip is a major disease that affects the healthy lifespan of a population. It is necessary to fully understand the patients’ conditions before a systematic treatment can be applied. However, a nationwide epidemiological study regarding hip OA has not yet been conducted in Japan. The present study examined the current status of patients with hip OA, including the disease etiology. Methods. This is a multiinstitutional study of new patients presenting with hip OA at the orthopedic outpatient clinics of 15 institutions in fi ve geographical areas of Japan. The collected data from each patient included the sex, age, treatment history for developmental dysplasia of the hip (DDH), the clinical score of the hip joints based on the Japanese Orthopaedic Association (JOA) scoring system, and the pelvic inclination according to anteroposterior radiographs. In addition, the etiology was determined from the following 17 options: primary OA, acetabular dysplasia, intragluteal dislocation, osteonecrosis, trauma, Perthes disease, slipped capital femoral epiphysis, infection, rheumatoid arthritis, ankylosing spondylitis, neuroarthropathy, endocrine diseases, metabolic diseases, hereditary bone diseases, synovial chondromatosis, generalized OA, and others. Results. There were a substantially larger number of female patients than male patients. This difference regarding sex was present in each generation. The mean age of the patients was 58 ± 14 years. The peak age at presentation was approximately 50 years. Most patients had no history of therapy for DDH. The older patients had lower gait and activities of daily living scores. The etiology was assessed to be acetabular dysplasia in most of the patients. A lower frequency of elderly patients demonstrated acetabular dysplasia. The patients who had a pelvic posterior inclination increased with increasing age. Conclusions. The patients with hip OA in Japan were unique in regard to age distribution, sexual heterogeneity, and disease

Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in primary knee osteoarthritis

OBJECTIVE To explore in a cross sectional study in patients with primary knee osteoarthritis (OA) the relations between body mass index (BMI), disease stage, and the concentrations of a putative joint fluid marker of type II collagen synthesis, procollagen II C-propeptide. PATIENTS AND METHODS The study included 142 patients with knee OA (median age 68, median BMI 24.1). OA was staged radiologically. The concentrations in synovial fluid of procollagen II C-propeptide were measured by a sandwich enzyme immunoassay. RESULTS Joint fluid concentrations of procollagen II C-propeptide were increased in knees with OA (median 3.7 ng/ml), compared with published reference values for knees in healthy adult volunteers (median 1.3 ng/ml). The concentrations of procollagen II C-propeptide were independently related to both OA stage and BMI (r s = 0.343, p < 0.0001 andr s = 0.253, p = 0.002, respectively). CONCLUSIONS Joint fluid concentrations of this putative marker of collagen II synthesis are high in early and mid-stage OA, but decrease in end stage disease. In addition and for the first time it was shown that the concentrations in synovial fluid of procollagen II C-propeptide increase with increasing BMI in primary knee OA. The increased joint fluid values of this marker in patients with primary knee OA and a high BMI, may reflect increased rates of collagen synthesis in their joint cartilage and could relate to the previously shown increased risk for disease progression in such patients.

Osteoarthritis hip joints in Japan: involvement of acetabular dysplasia

BackgroundWe conducted a nationwide epidemiologic study regarding hip osteoarthritis (OA) in Japan, and a previous report found these patients to be unique in comparison to Caucasians. This report focused on the data regarding each hip joint, and the involvement of acetabular dysplasia with hip OA was analyzed.MethodsSeven hundred twenty OA hips were examined. Sixty-five joints with osteonecrosis of the femoral head and 215 non-OA contralateral joints of the unilateral patients were examined as controls. The revised system of stage classification for hip OA of the Japanese Orthopedic Association (JOA) was used according to the reproducibility in order to ensure reliable data from the multiple institutions. The acetabular dysplasia indexes were also chosen according to the reproducibility and measured in the radiograph of bilateral hip joints. The clinical score was assessed using the JOA scoring system. The relative risk of the grade of acetabular dysplasia indexes for hip OA was calculated as the odds ratio and the 95% confidence interval.ResultsThe stage of the OA joints deteriorated with increasing age. The clinical scores also decreased. The grade of the acetabular dysplasia indexes of the OA joints was significantly higher than that of the control joints. Each index of acetabular dysplasia demonstrated significantly increased odds ratios for hip OA. Among the OA joints, the deterioration of the OA stage was found to be significantly associated with an increasing grade of acetabular dysplasia. The odds ratio for OA deterioration in the acetabular dysplasia index was also obtained. The joints of females tended to have a higher grade and prevalence of acetabular dysplasia than those of males.ConclusionsThese findings confirmed a high prevalence of acetabular dysplasia in hip OA joints in Japan. Acetabular dysplasia was one of the most important factors associated with hip OA.