Moritoshi Furu

Expression of the p16INK4A Gene Is Associated Closely with Senescence of Human Mesenchymal Stem Cells and Is Potentially Silenced by DNA Methylation During In Vitro Expansion

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A‐positive cells were Ki67‐negative and senescence associated β‐galactosidase‐positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty‐five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase.

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO(2)) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO(2)) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

Comprehensive microRNA Analysis Identifies miR-24 and miR-125a-5p as Plasma Biomarkers for Rheumatoid Arthritis

MicroRNAs (miRNAs) are present in human plasma and known as a non-invasive biomarker for cancer detection. Our study was designed to identify plasma miRNAs specific for rheumatoid arthritis (RA) by a comprehensive array approach. We performed a systematic, array-based miRNA analysis on plasma samples from three RA patients and three healthy controls (HCs). Plasma miRNAs with more than four times change or with significant (P<0.05) change in expression, or detectable only in RA plasma, were confirmed with plasma from eight RA patients and eight HCs using real-time quantitative PCR. Consistently detectable miRNAs that were significantly different between RA patients and HCs were chosen for further validation with 102 RA patients and 104 HCs. The area under curves (AUC) were calculated after plotting the receiver operating characteristic (ROC) curves. To determine if these miRNAs are specific for RA, the concentrations of these miRNAs were analyzed in 24 patients with osteoarthritis (OA), and 11 patients with systemic lupus erythematosus (SLE). The array analysis and the subsequent confirmation in larger patient cohort identified significant alterations in plasma levels of seven miRNAs. The highest AUC was found for miR-125a-5p, followed in order by miR-24 and miR-26a. Multivariable logistic regression analysis showed that miR-24, miR-30a-5p, and miR-125a-5p were crucial factors for making detection model of RA and provided a formula for Estimated Probability of RA by plasma MiRNA (ePRAM), employing miR-24, miR-30a-5p and miR-125a-5p, which showed increased diagnostic accuracy (AUC: 0.89). The level of miR-24, miR-125a-5p, and ePRAM in OA and SLE patients were lower than that in RA. There was no significant difference in detection for anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative RA patients. These results suggest that the plasma concentrations of miR-24 and miR-125a-5p, and ePRAM are potential diagnostic markers of RA even if patients were ACPA-negative.

Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease

T cells that mediate autoimmune diseases such as rheumatoid arthritis (RA) are difficult to characterize because they are likely to be deleted or inactivated in the thymus if the self antigens they recognize are ubiquitously expressed. One way to obtain and analyze these autoimmune T cells is to alter T cell receptor (TCR) signaling in developing T cells to change their sensitivity to thymic negative selection, thereby allowing their thymic production. From mice thus engineered to generate T cells mediating autoimmune arthritis, we isolated arthritogenic TCRs and characterized the self antigens they recognized. One of them was the ubiquitously expressed 60S ribosomal protein L23a (RPL23A), with which T cells and autoantibodies from RA patients reacted. This strategy may improve our understanding of the underlying drivers of autoimmunity. In a mouse model of rheumatoid arthritis, autoimmune T cells recognize a protein from the ribosome. Finding the targets of T cells gone bad Autoimmune diseases such as rheumatoid arthritis can result when the immune system attacks its own body. If we could identify the specific proteins targeted by autoimmune T cells, we might then be able to block this interaction, which might be useful therapeutically. Ito et al. identified one such target in mice that develop a disease similar to rheumatoid arthritis. Disease-causing T cells recognized a protein that is part of the ribosome, a large protein complex that catalyzes protein synthesis. They also found T cells specific for this protein in people with rheumatoid arthritis. Science, this issue p. 363

Inhibition of miR-92a enhances fracture healing via promoting angiogenesis in a model of stabilized fracture in young mice.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes. Although no miRNA has been reported to play an important role in the regulation of fracture healing, several miRNAs control key elements in tissue repair processes such as inflammation, hypoxia response, angiogenesis, stem cell differentiation, osteogenesis, and chondrogenesis. We compared the plasma concentrations of 134 miRNAs in 4 patients with trochanteric fractures and 4 healthy controls (HCs), and the levels of six miRNAs were dysregulated. Among these miRNAs, miR‐92a levels were significantly decreased 24 hours after fracture, compared to HCs. In patients with a trochanteric fracture or a lumbar compression fracture, the plasma concentrations of miR‐92a were lower on days 7 and 14, but had recovered on day 21 after the surgery or injury. To determine whether systemic downregulation of miR‐92a can modulate fracture healing, we administered antimir‐92a, designed using locked nucleic acid technology to inhibit miR‐92a, to mice with a femoral fracture. Systemic administration of antimir‐92a twice a week increased the callus volume and enhanced fracture healing. Enhancement of fracture healing was also observed after local administration of antimir‐92a. Neovascularization was increased in mice treated with antimir‐92a. These results suggest that plasma miR‐92a plays a crucial role in bone fracture healing in human and that inhibition of miR‐92a enhances fracture healing through angiogenesis and has therapeutic potential for bone repair.

MicroRNA-451 Down-Regulates Neutrophil Chemotaxis via p38 MAPK

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate the activity of target messenger RNAs (mRNAs) and cellular processes. MicroRNA‐451 (miR‐451) is one of the miRNAs that is conserved perfectly among vertebrates, and it regulates cell proliferation, invasion, and apoptosis in tumors. However, the role of miR‐451 in autoimmune arthritis is unknown. This study was undertaken to identify the role of miR‐451 in autoimmune arthritis.

Kinematic alignment produces near-normal knee motion but increases contact stress after total knee arthroplasty: A case study on a single implant design

BACKGROUND Kinematically aligned total knee arthroplasty (TKA) is of increasing interest because this method might improve postoperative patient satisfaction. In kinematic alignment the femoral component is implanted in a slightly more valgus and internally rotated position, and the tibial component is implanted in a slightly more varus and internally rotated position, than in mechanical alignment. However, the biomechanics of kinematically aligned TKA remain largely unknown. The aim of this study was to compare the kinematics and contact stresses of mechanically and kinematically aligned TKAs. METHODS A musculoskeletal computer simulation was used to determine the effects of mechanically or kinematically aligned TKA. Knee kinematics were examined for mechanically aligned, kinematically aligned, and kinematically aligned outlier models. Patellofemoral and tibiofemoral contact forces were measured using finite element analysis. RESULTS Greater femoral rollback and more external rotation of the femoral component were observed with kinematically aligned TKA than mechanically aligned TKA. However, patellofemoral and tibiofemoral contact stresses were increased in kinematically aligned TKA. CONCLUSIONS These findings suggest that kinematically aligned TKA produces near-normal knee kinematics, but that concerns for long-term outcome might arise because of high contact stresses.

A Distinct Human CD4+ T Cell Subset That Secretes CXCL13 in Rheumatoid Synovium

OBJECTIVE A subset of CD4+ T cells in the synovium of patients with rheumatoid arthritis (RA) produce CXCL13, a chemokine that is crucial for the formation of germinal centers. This study was undertaken to determine the relevance of this population to known subsets of T helper cells and to proinflammatory cytokines, and how these cells are generated. METHODS The expression of Th markers and CXCL13 by CD4+ T cells in RA synovium and the involvement of proinflammatory cytokines in CXCL13 production were assessed. We also investigated whether CXCL13+CD4+ T cells could be newly induced. RESULTS CXCL13+CD4+ T cells in RA synovium were negative for interferon-γ (IFNγ), interleukin-4 (IL-4), IL-17, FoxP3, and CXCR5 and expressed low levels of inducible T cell costimulator, indicating that this population is a distinct human CD4 subset. T cell receptor (TCR) stimulation of CD4+ T cells, obtained from RA synovium with low expression of CXCL13, promptly induced CXCL13 production and addition of proinflammatory cytokines supported the long-term production of CXCL13. These findings indicate that CXCL13-producing CD4+ T cells can be in a memory state ready to be reactivated upon TCR stimulation and that proinflammatory cytokines are involved in persistent CXCL13 production. TCR stimulation of CD4+ T cells from the blood of healthy volunteers, together with proinflammatory cytokine supplementation, induced a population that produced CXCL13, but not IFNγ. Synovial T cells recruited CXCR5+ cells in a CXCL13-dependent manner. CONCLUSION CXCL13-producing CD4+ T cells induced in RA synovium may play a role in the recruitment of CXCR5+ cells, such as B cells and circulating follicular helper T cells, and in ectopic lymphoid neogenesis at sites of inflammation.

Discordance and accordance between patient’s and physician’s assessments in rheumatoid arthritis

Objectives: The American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) remission criteria for rheumatoid arthritis (RA) are more stringent than index-based criteria, making it more difficult to achieve a patient’s global assessment (PGA) than an evaluator’s global assessment (EGA). We investigated the reason for the discrepancy between the PGA and the EGA in a Japanese clinical cohort. Method: We assessed clinical and laboratory variables in our clinical cohort. The frequency of remission achievement according to the ACR/EULAR remission criteria and predictors of the discrepancy between the PGA and EGA were analysed. Results: Of 370 patients with RA, 89 fulfilled PGA criteria and 167 patients fulfilled EGA criteria. The PGA was highly correlated with the visual analogue scale (VAS) pain score and non-inflammatory variables including Steinbrocker class and the Health Assessment Questionnaire Disability Index (HAQ-DI). Conversely, inflammatory variables, including swollen joint count (SJC), tender joint count (TJC), and C-reactive protein (CRP) levels, were significantly associated with the EGA. The main predictors of the discrepancy between the PGA and the EGA were patient’s VAS pain score, SJC, and functional disability. Conclusions: Increased pain and functional disability led to a discrepancy towards a worse PGA than EGA, whereas increased SJC led to an accordance towards a worse EGA.

Malrotated Tibial Component Increases Medial Collateral Ligament Tension in Total Knee Arthroplasty

Malrotation of the tibial component can lead to complications after total knee arthroplasty (TKA). Despite reports of internal rotation being associated with more severe pain or stiffness than external rotation, the biomechanical reasons remain largely unknown. We used a computer simulation model and evaluated traction forces in the lateral collateral ligament (LCL) and medial collateral ligament (MCL) with a malrotated tibial component during squatting. We also examined tibiofemoral and patellofemoral contact forces and stresses under similar conditions. A dynamic musculoskeletal knee model was simulated in three different constrained tibial geometries with a prototype component. The testing conditions were changed between 15° external and 15° internal rotation of the tibial component. With internal rotation of the tibial component, the MCL force increased progressively; the LCL force also increased, but only up to less than half of the MCL force values. A higher degree of constraint of the tibial component was associated with greater femoral rotational movement and higher MCL forces. The tibiofemoral and patellofemoral contact forces were not influenced by malrotation of the tibial component, but the contact stresses increased because of decreased contact area. This altered loading condition could cause patient complaints and polyethylene problems after TKA.