Hiraku Motomura

PDGF Receptor β Is a Potent Regulator of Mesenchymal Stromal Cell Function

Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet‐derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)β gene by Cre‐loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF‐A, ‐B, ‐C, and ‐D as well as PDGFRα and β was detected. Depletion of PDGFRβ in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT‐PCR. PDGF‐BB, but not PDGF‐AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF‐BB were eliminated by depletion of PDGFRβ in MSCs except that PDGF‐BB still suppressed osterix expression in PDGFRβ‐depleted MSCs. Depletion of PDGFRβ significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRβ gene deletion, we showed that PDGFRβ signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRα on the osteogenic differentiation were very subtle. PDGFRβ could represent an important target for guided tissue regeneration or tissue engineering of bone.

BMP-2 prevents apoptosis of the N1511 chondrocytic cell line through PI3K/Akt-mediated NF-κB activation

The signal transduction pathway by which bone morphogenetic protein-2 (BMP-2) regulates apoptosis in chondrocytes remains largely unknown. We investigated the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt-mediated NF-κB activation by BMP-2 stimulation in the modulation of this antiapoptotic process in a chondrocytic cell line, N1511. BMP-2 prevented apoptosis through the inhibition of caspase-3 and -9 and an increase in Bcl-xL expression, and this antiapoptotic effect was inhibited by Noggin. Not only was NF-κB p65 activated transiently in the early phase (5–15 min) after treatment with BMP-2 but p65 at serine 536 was phosphorylated from 5 min as well. Akt was rapidly phosphorylated in response to BMP-2 treatment; however, the inhibition of PI3K by Wortmannin markedly reduced the phosphorylation of Akt by BMP-2. Wortmannin also decreased the NF-κB transcriptional activity that was up-regulated by BMP-2. Thus, BMP-2-induced NF-κB activation is mediated by PI3K/Akt signaling. Wortmannin treatment inhibited the antiapoptotic effect of BMP-2. These data indicate that BMP-2 can utilize a new signal transduction pathway in the NF-κB activation system, which plays a crucial role in the survival of the N1511 chondrocytic cell line.

Cartilage intermediate layer protein promotes lumbar disc degeneration

Lumbar disc disease (LDD) is one of the most common musculoskeletal disorders, and accompanies intervertebral disc degeneration. CILP encodes cartilage intermediate layer protein, which is highly associated with LDD. Moreover, CILP inhibits transcriptional activation of cartilage matrix genes in nucleus pulposus (NP) cells in vitro by binding to TGF-β1 and inhibiting the phosphorylation of Smads. However, the aetiology and mechanism of pathogenesis of LDD in vivo are unknown. To demonstrate the role of CILP in LDD in vivo, we generated transgenic mice that express CILP specifically in the intervertebral disc tissues and assessed whether CILP exacerbates disc degeneration. Degeneration of the intervertebral discs was assessed using magnetic resonance imaging (MRI) and histology. The level of phosphorylation of Smad2/3 in intervertebral discs was measured to determine whether overexpressed CILP suppressed TGF-beta signalling. Although the macroscopic skeletal phenotype of transgenic mice appeared normal, histological findings revealed significant degeneration of lumbar discs. MRI analysis of the lumbar intervertebral discs indicated a significantly lower signal intensity of the nucleus pulposus where CILP was overexpressed. Intervertebral disc degeneration was also observed. The number of phosphorylation of Smad2/3 immuno-positive cells in the NP significantly was decreased in CILP transgenic mice compared with normal mice. In summary, overexpression of CILP in the NP promotes disc degeneration, indicating that CILP plays a direct role in the pathogenesis of LDD.

Inhibitory effect of tacrolimus on progression of joint damage in patients with rheumatoid arthritis

To examine the inhibitory effect of tacrolimus on radiographic joint damage in patients with rheumatoid arthritis (RA).

Radiographic changes and factors associated with subsequent progression of damage in weight-bearing joints of patients with rheumatoid arthritis under TNF-blocking therapies—three-year observational study

Abstract Objectives: The long-term effects of tumor necrosis factor (TNF)-blocking therapies on weight-bearing joints in patients with rheumatoid arthritis (RA) have not been fully characterized. The purpose of this study was to assess the radiographic changes of weight-bearing joints in patients with RA during 3-year of TNF-blocking therapies and to identify factors related to the progression of joint damage. Methods: Changes in clinical variables and radiological findings in 243 weight-bearing joints (63 hips, 54 knees, 71 ankles, and 55 subtalar joints) in 38 consecutive patients were investigated during three years of treatment with TNF-blocking agents. Multivariate logistic regression analysis was used to identify risk factors for the progression of weight-bearing joint damage. Results: Seventeen (14.5%) of proximal weight-bearing joints (hips and knees) showed apparent radiographic progression during three years of treatment, whereas none of the proximal weight-bearing joints showed radiographic evidence of improvement or repair. In contrast, distal weight-bearing joints (ankle and subtalar joints) displayed radiographic progression and improvement in 20 (15.9%) and 8 (6.3%) joints, respectively. Multivariate logistic analysis for proximal weight-bearing joints identified the baseline Larsen grade (p < 0.001, OR:24.85, 95%CI: 5.07–121.79) and disease activity at one year after treatment (p = 0.003, OR:3.34, 95%CI:1.50–7.46) as independent factors associated with the progression of joint damage. On the other hand, multivariate analysis for distal weight-bearing joints identified disease activity at one year after treatment (p < 0.001, OR:2.13, 95%CI:1.43–3.18) as an independent factor related to the progression of damage. Conclusions: Baseline Larsen grade was strongly associated with the progression of damage in the proximal weight-bearing joints. Disease activity after treatment was an independent factor for progression of damage in proximal and distal weight-bearing joints. Early treatment with TNF-blocking agents and tight control of disease activity are necessary to prevent the progression of damage of the weight-bearing joints.

Long-term clinical and radiographic results of cementless total hip arthroplasty for patients with rheumatoid arthritis: minimal 10-year follow-up

Abstract Objectives. The aim of this study was to clarify the long-term clinical and radiographic results of cementless total hip arthroplasty (THA) for patients with rheumatoid arthritis (RA). Methods. Twenty-eight total hip arthroplasties in 24 patients with a diagnosis of RA were performed from October 1992 to October 1996. All components were titanium alloy with a circumferential porous coating. Six patients (six hips) died before the 10-year follow-up, and one patient (one hip) was lost to follow-up, leaving 21 joints of 17 patients for review at a minimum 10-year follow-up after surgery. There were 3 men and 14 women with an average age of 55.0 years. The average duration of RA at the time of the operation was 12.6 years, and the average follow-up period was 12.2 years. We evaluated the Japanese Orthopaedic Association (JOA) hip scores, radiographic changes and survivor rates of components. Results. Compared with the preoperative JOA hip scores, there was significant improvement in the postoperative scores. Spot welds consistent with bone ingrowth were identified in 95.0% of the femoral components. No femoral components showed radiographic loosening or required revision for aseptic loosening, but two acetabular revisions were performed because of aseptic loosening. The 14-year survivor rates of the stem and cup with the end point of loosening were 100% and 88.2%, respectively. Conclusions. Cementless THA with this component design in patients with RA appears to be a promising treatment.

Myxoid Liposarcoma-Associated EWSR1-DDIT3 Selectively Represses Osteoblastic and Chondrocytic Transcription in Multipotent Mesenchymal Cells

Background Liposarcomas are the most common class of soft tissue sarcomas, and myxoid liposarcoma is the second most common liposarcoma. EWSR1-DDIT3 is a chimeric fusion protein generated by the myxoid liposarcoma-specific chromosomal translocation t(12;22)(q13;q12). Current studies indicate that multipotent mesenchymal cells are the origin of sarcomas. The mechanism whereby EWSR1-DDIT3 contributes to the phenotypic selection of target cells during oncogenic transformation remains to be elucidated. Methodology/Principal Findings Reporter assays showed that the EWSR1-DDIT3 myxoid liposarcoma fusion protein, but not its wild-type counterparts EWSR1 and DDIT3, selectively repressed the transcriptional activity of cell lineage-specific marker genes in multipotent mesenchymal C3H10T1/2 cells. Specifically, the osteoblastic marker Opn promoter and chondrocytic marker Col11a2 promoter were repressed, while the adipocytic marker Ppar-γ2 promoter was not affected. Mutation analyses, transient ChIP assays, and treatment of cells with trichostatin A (a potent inhibitor of histone deacetylases) or 5-Aza-2′-deoxycytidine (a methylation-resistant cytosine homolog) revealed the possible molecular mechanisms underlying the above-mentioned selective transcriptional repression. The first is a genetic action of the EWSR1-DDIT3 fusion protein, which results in binding to the functional C/EBP site within Opn and Col11a2 promoters through interaction of its DNA-binding domain and subsequent interference with endogenous C/EBPβ function. Another possible mechanism is an epigenetic action of EWSR1-DDIT3, which enhances histone deacetylation, DNA methylation, and histone H3K9 trimethylation at the transcriptional repression site. We hypothesize that EWSR1-DDIT3-mediated transcriptional regulation may modulate the target cell lineage through target gene-specific genetic and epigenetic conversions. Conclusions/Significance This study elucidates the molecular mechanisms underlying EWSR1-DDIT3 fusion protein-mediated phenotypic selection of putative target multipotent mesenchymal cells during myxoid liposarcoma development. A better understanding of this process is fundamental to the elucidation of possible direct lineage reprogramming in oncogenic sarcoma transformation mediated by fusion proteins.

Lumbar spine surgery in patients with rheumatoid arthritis (RA): what affects the outcomes?

BACKGROUND CONTEXT Although the cervical spine is only occasionally involved in rheumatoid arthritis (RA), involvement of the lumbar spine is even less common. A few reports on lumbar spinal stenosis in patients with RA have appeared. Although disc space narrowing occurs in aging, postoperative adjacent segment disease (ASD) in patients with RA has not been subject to much analysis. PURPOSE The objective of this study was to investigate differences in ASD and clinical outcomes between lumbar spinal decompression with and without fusion in patients with RA. STUDY DESIGN/SETTING This is a retrospective comparative study. PATIENT SAMPLE A total of 52 patients with RA who underwent surgery for lumbar spinal disorders were included. Twenty-seven patients underwent decompression surgery with fusion and 25 underwent decompression surgery alone. OUTCOME MEASURES Intervertebral disc space narrowing and spondylolisthesis of the segment immediately cranial to the surgical site were measured using a three-dimensional volume rendering software. Pre- and postoperative evaluation of RA activity and Japanese Orthopaedic Association (JOA) scores were conducted. MATERIALS AND METHODS All patients had preoperative and annual postoperative lumbar radiographs and were followed up for a mean of 5.1 years (range 3.5-10.9 years). Pre- and postoperative (2 years after surgery) JOA scores were recorded and any postoperative complications were investigated. Degrees of intervertebral disc narrowing and spondylolisthesis at the adjacent levels were evaluated on radiographs and were compared between the two groups. Analysis was performed to look for any correlation between ASD and RA disease activities. RESULTS Postoperative JOA scores were significantly improved in both groups. The rate of revision surgery was significantly higher in the fusion group than that in the non-fusion group. The rate of ASD was significantly greater in the fusion group than that in the non-fusion group at the final follow-up examination. Both matrix metalloproteinase 3 (MMP-3) and the 28-joint disease activity score incorporating C-reactive protein levels (DAS28-CRP) were significantly associated with the incidence and severity of ASD. CONCLUSIONS Adjacent segment disease and the need for revision surgery were significantly higher in the fusion group than those in the non-fusion group. A preoperative high MMP-3 and DAS28-CRP are likely to be associated with postoperative ASD.

A selective inhibition of c-Fos/activator protein-1 as a potential therapeutic target for intervertebral disc degeneration and associated pain

Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1β-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.

FRI0508 Correlation of power doppler ultrasonographic findings with site-matched histopathology of the synovial tissue

Background Ultrasonography (US) has been recently established as an imaging modality to evaluate inflamed joints of patients with rheumatoid arthritis (RA). It is reported that power doppler ultrasonography (PDUS) is capable of directly visualizing and quantifying synovial inflammation [1]. However, detailed correlation between the PDUS findings and histopathological findings of RA synovial tissues, such as accumulation of various inflammatory cells, neovasculogenesis, and synovial hyperplasia, still remains unclear. Objectives To assess the significance of PDUS in the evaluation of synovial lesion of the knee joint by comparing the PDUS findings with site-matched histopathological findings. Methods We studied 23 patients who were undergoing arthroplasty of the knee joint because of RA or osteoarthritis (OA). Predetermined sites of the knee joint were examined with US before arthroplasty. Synovitis was classified semiquantitatively with grey scale (GS) and PDUS findings. PD signal was graded as 0 (normal), 1 (minor), 2 (moderate), and 3 (major) [2]. Synovial tissues were obtained during arthroplasty from the corresponding sites evaluated by the US. Histopathology of the synovial tissue was evaluated by haematoxylin and eosin staining and immunohistochemical staining with factor VIII. The degree of inflammatory cell infiltrates, synovial lining layer thickness and vascularity was graded as 0 (normal), 1 (mild change), 2 (moderate change), and 3 (severe change) [3]. Results Positive PDUS (grade 2 or 3) was found in 11 of 19 knee joints in patients with RA, but was not observed in all patients with OA. We compared imaging findings of preoperative PDUS with histopathological findings of the synovial specimens. We found that the PDUS findings were mostly correlated with each histopathological grade of synovitis and the vascularity showed the highest correlation with the PDUS findings (r=0.541, P<0.01) as expected. However, the site-matched comparison indicated that the blood vessels, possibly low-velocity blood flow, were not apparently detected by the PDUS. Thus, the sensitivity and specificity of PDUS findings for histopathological vascularity were 91% and 58%, respectively. Conclusions We showed that positive findings of PDUS were closely associated with histopathological findings of active synovitis. However, our results suggest that PDUS is not sufficiently powerful in evaluating blood vessels with low-velocity blood flow in the synovial tissue. References Naredo E, et al. Ann Rheum Dis. 2008; 67:1017-22. Hammer HB, et al. Ann Rheum Dis. 2011; 70:1995-8. Takase K, Clin Exp Rheumatol. 2012; 30: 85-92. Disclosure of Interest None Declared