Differential proteome analysis in adolescent idiopathic scoliosis patients with thoracolumbar/lumbar curvatures.

BACKGROUND: Although the pathogenesis of adolescent idiopathic scoliosis (AIS) remains unclear, there are little evidences of the pathogenesis in patients with thoracolumbar/lumbar AIS. The purpose of this study was to identify proteins or proteomes that may be causally related to the pathogenesis of AIS with structured thoracolumbar/lumbar curvature using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: A total of 20 control volunteers and 61 AIS in patients with thoracolumbar/lumbar curvature were included. First, the plasma samples of each five AIS with pure thoracolumbar/lumbar curvature and control samples were subjected to 2D-DIGE analysis. Protein spots that were expressed differently by the AIS and control groups were selected and identified by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis. To characterize the differently-expressed proteins in AIS patients, we performed functional pathway analysis using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) system. Additionally, the proteins were compared between control and AIS using western blotting. Lastly, prospectively collected 15 control and 41 AIS with thoracolumbar/lumbar curvature samples were compared to the differentially expressed proteins. RESULTS: A total of 3862 ± 137 spots were detected, of which 11 spots met the criteria when compared with controls. Nine proteins were identified by nanoLC-MS/MS. Functional analysis showed the association of the proteins in AIS patients with blood coagulation using the PANTHER system. Of the proteins, vitamin D binding protein (DBP) significantly correlated with Cobb angle in thoracolumbar/lumbar curvatures. DBP expression of the prospectively collected AIS samples were significantly higher than those of controls (P < 0.05). CONCLUSIONS: This study suggests that DBP and several coagulation-related proteins may play a role in the pathogenesis of AIS. DBP appears to be a marker of severity of AIS with thoracolumbar/lumbar curvature.

A selective c-Fos/AP-1 inhibitor prevents cartilage destruction and subsequent osteophyte formation.

The objective of the present study is to demonstrate that a newly developed selective c-Fos/activator protein (AP)-1 inhibitor, T-5224, inhibits the expression of matrix metalloproteinases (MMPs) in human articular chondrocytes, and prevents cartilage destruction in an osteoarthritis (OA)-induced mouse model. First, we examined the effect of T-5224 on MMP and inflammatory cytokine expression by real-time polymerase chain reaction in human articular chondrocytes. We created an OA model by destabilization of the medial meniscus (DMM) in mice. T-5224 was orally administered once a day and the OA pathology was assessed by histological, immunohistochemical, and micro-computed tomography (CT) analyses. T-5224 inhibited the mRNA expression levels of MMP-1, 3, and 13, and interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6 in IL-1-stimulated human chondrocytes. Oral administration of T-5224 to OA-induced mice prevented cartilage destruction. The histological scores for OA were significantly better in the T-5224-treated group than the vehicle-treated group. Type X collagen and MMP-13 were not increased in the T-5224-treated group by immunohistochemical staining. Micro-CT analysis showed mild but apparent osteophyte development in the femoral condyle and antero-medial aspect of the tibia in the vehicle-treated group but not in the T-5224-treated group. Taken together, specific inhibition of c-Fos/AP-1 and the resulting inhibition of the transactivation of a broad spectrum of downstream MMPs, along with inflammatory cytokines, effectively prevented cartilage destruction and osteophyte formation.

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-1ß-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.

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.

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.

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

OBJECTIVE: Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. MATERIALS AND METHODS: ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. RESULTS: The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. CONCLUSION: These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

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-ß1 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.

AIM: To examine the inhibitory effect of tacrolimus on radiographic joint damage in patients with rheumatoid arthritis (RA). METHODS: Thirty-eight patients with RA resistant or intolerant to conventional disease-modifying anti-rheumatic drugs were administered tacrolimus and analyzed retrospectively. Disease activity and clinical response were evaluated by Disease Activity Score in 28 joints and C-reactive protein (DAS28-CRP) and European League Against Rheumatism (EULAR) response criteria. The progression of joint destruction was evaluated by an estimated yearly change in modified Total Sharp Score (mTSS). RESULTS: Good or moderate response rate according to EULAR response criteria was seen in 63.2%, 63.2%, 73.7% and 65.8% of patients at 3, 6, 12, and 24 months, respectively. The rate of patients with low disease activity or remission reached 47.3% and 50.0% at 12 and 24 months, respectively. Progression of joint damage, evaluated as yearly change in mTSS (ΔmTSS), significantly decreased from 11.4 at baseline to 2.63 in the first year and 0.69 in the second year of tacrolimus treatment. CONCLUSION: These findings suggest tacrolimus has the potential to inhibit progression of joint damage in established RA.

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

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/EBPß 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.

Radiographic progression in weight-bearing joints of patients with rheumatoid arthritis after TNF-blocking therapies.

The aim of the present study was to assess the influence of tumor necrosis factor (TNF)-blocking therapies on weight-bearing joints in patients with rheumatoid arthritis. Changes in clinical variables and radiological findings in 213 weight-bearing joints (69 hip joints, 63 knee joints, and 81 ankle joints) of 42 consecutive patients were investigated at baseline and at 1 year of TNF-blocking therapies. Structural damage to the weight-bearing joints was assessed using the Larsen scoring method. Detailed comparisons of the sizes and locations of erosions were performed for each set of radiographs of the respective joints. Assessment of radiographs of the 213 weight-bearing joints indicated progression of the Larsen grade in eight joints. Another five joints without Larsen grade progression showed apparent radiographic progression of joint damage based on increases in bony erosions. Overall, 13 joints (6%) of eight patients (19%) showed progression of joint damage after 1 year of TNF-blocking therapies. Analysis of each baseline grade indicated that radiographic progression of joint damage was inhibited in most grade 0-II joints. On the other hand, all hip and knee joints with pre-existing damage of grade III/IV showed apparent progression even in patients with good response. The results further suggested that radiographic progression may occur in less damaged joints when the patients were non-responders to the therapy. Among the weight-bearing joints, ankle joints showed different radiographic behavior and four ankle joints displayed improvement of radiographic damage. Early initiation of anti-TNF therapy should be necessary especially when the patients are starting to show early structural damage in weight-bearing joints.

PDGF receptor beta 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)beta 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 PDGFRalpha and beta was detected. Depletion of PDGFRbeta 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 PDGFRbeta in MSCs except that PDGF-BB still suppressed osterix expression in PDGFRbeta-depleted MSCs. Depletion of PDGFRbeta significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRbeta gene deletion, we showed that PDGFRbeta signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRalpha on the osteogenic differentiation were very subtle. PDGFRbeta could represent an important target for guided tissue regeneration or tissue engineering of bone.

Gas6, a new regulator of chondrogenic differentiation from mesenchymal cells.

The mesenchymal cell line C3H10T1/2 can be preferentially induced toward chondrogenesis by culturing as a micromass in the presence of bone morphogenetic protein 2. To screen new regulator genes for chondrogenic differentiation, we performed differential display polymerase chain reaction and identified growth arrest-specific 6 (Gas6) as a gene that was clearly downregulated by this induction of chondrogenic differentiation. Blockage of Gas6 mRNA expression by siRNA remarkably enhanced the chondrogenic differentiation, while stimulation with recombinant Gas6 inhibited the mRNA expressions of type II collagen (Col2a1) and aggrecan. Gas6 signaling activated the phosphorylation of ERK1/2, SAPK/JNK, and Akt, but not p38 MAPK. These results suggest that Gas6 negatively regulates chondrogenic differentiation, at least through the MAPK pathway.

PEA3 cooperates with c-Jun in regulation of HER2/neu transcription.

HER2/neu overexpressing breast tumors exhibit an increase in polyomavirus enhancer activator 3 (PEA3) expression. We examined the relationship between HER2/neu transcriptional activation and PEA3 in cooperation with c-Jun. HER2/neu promoter activity was decreased by deleting PEA3 binding site, and was downregulated when the PEA3 binding site was mutated. PEA3 and c-Jun each weakly enhanced luciferase expression of the HER2/neu promoter. However, the HER2/neu promoter response to PEA3 was considerably enhanced by c-Jun. Thus, we examined the interaction of PEA3 with c-Jun by the two-hybrid system, the transcriptional activity of PEA3 was specifically enhanced by c-Jun. When PEA3, c-Jun and coactivator p300 were cotransfected in MCF7 cells, the transcriptional activity of HER2/neu was increased by up to 20-fold. PEA3 and c-Jun-induced transcription of HER2/neu promoter was repressed by cotransfection of the dominant negative of p300. These results suggest that PEA3 and c-Jun stimulated synergistically the HER2/neu gene transcription with p300.

BMP-2 prevents apoptosis of the N1511 chondrocytic cell line through PI3K/Akt-mediated NF-kappaB 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-kappaB 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-kappaB 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-kappaB transcriptional activity that was up-regulated by BMP-2. Thus, BMP-2-induced NF-kappaB 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-kappaB activation system, which plays a crucial role in the survival of the N1511 chondrocytic cell line.