N. Hiraoka

Osteoblasts derived from osteophytes produce interleukin-6, interleukin-8, and matrix metalloproteinase-13 in osteoarthritis

To clarify the significance of the osteophytes that appear during the progression of osteoarthritis (OA), we investigated the expression of inflammatory cytokines and proteases in osteoblasts from osteophytes. We also examined the influence of mechanical stress loading on osteoblasts on the expression of inflammatory cytokines and proteases. Osteoblasts were isolated from osteophytes in 19 patients diagnosed with knee OA and from subchondral bone in 4 patients diagnosed with femoral neck fracture. Messenger RNA expression and protein production of inflammatory cytokines and proteases were analyzed using real-time RT-PCR and ELISA, respectively. To examine the effects of mechanical loading, continuous hydrostatic pressure was applied to the osteoblasts. We determined the mRNA expression and protein production of IL-6, IL-8, and MMP-13, which are involved in the progression of OA, were increased in the osteophytes. Additionally, when OA pathological conditions were simulated by applying a nonphysiological mechanical stress load, the gene expression of IL-6 and IL-8 increased. Our results suggested that nonphysiological mechanical stress may induce the expression of biological factors in the osteophytes and is involved in OA progression. By controlling the expression of these genes in the osteophytes, the progression of cartilage degeneration in OA may be reduced, suggesting a new treatment strategy for OA.

Hyperthermia for the treatment of articular cartilage with osteoarthritis

Osteoarthritis (OA) is one of the most frequent musculoskeletal disorders in the elderly population. OA is characterised by a gradual loss of extracellular matrix in the articular cartilage of joints. OA can only be managed by artificial joint replacement when joint destruction becomes severe. Therefore, it is preferable to administer conservative therapy that is easy, simple and effective in inhibiting OA progression at the early stage. Heat shock protein 70 (Hsp70) has a protective effect on the cartilage and inhibits the apoptosis of chondrocytes. Heat stimulation by microwave to the joints can increase Hsp70 expression in chondrocytes, and at the same time, Hsp70 expression partially enhances matrix metabolism of the cartilage. These findings suggest that hyperthermia can be positively applied to the treatment of OA. Hyperthermia is therefore expected to be an inexpensive and less-invasive conservative therapy for OA.

Intra-Articular Injection of Hyaluronan Restores the Aberrant Expression of Matrix Metalloproteinase-13 in Osteoarthritic Subchondral Bone

Subchondral bone is a candidate for treatment of osteoarthritis (OA). We investigated the effects of intra‐articular injection of hyaluronan (IAI‐HA) on subchondral bone in rabbit OA model. OA was induced by anterior cruciate ligament transection, with some rabbits receiving IAI‐HA. OA was graded morphologically, and expression of mRNA was assessed by real‐time RT‐PCR. Tissue sections were stained with hyaluronan‐binding protein, and penetration of fluorescent hyaluronan was assessed. The in vitro inhibitory effect of hyaluronan on MMP‐13 was analyzed in human osteoarthritic subchondral bone osteoblasts (OA Ob) by real‐time RT‐PCR and ELISA. Binding of hyaluronan to OA Ob via CD44 was assessed by immunofluorescence cytochemistry. Expression of MMP‐13 and IL‐6 mRNA in cartilage and subchondral bone, and morphological OA grade, increased over time. IAI‐HA ameliorated the OA grade and selectively suppressed MMP‐13 mRNA in subchondral bone. IAI‐HA enhanced the hyaluronan staining of subchondral bone marrow cells and osteocyte lacunae. Fluorescence was observed in the subchondral bone marrow space. In OA Ob, hyaluronan reduced the expression and production of MMP‐13, and anti‐CD44 antibody blocked hyaluronan binding to OA Ob. These findings indicate that regulation of MMP‐13 in subchondral bone may be a critical mechanism during IAI‐HA.

Asporin and transforming growth factor-β gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis

BackgroundTo clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-β1 (TGF-β1), TGF-β2, TGF-β3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism.MethodsOsteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-β1, TGF-β2, TGF-β3, and Runx2 was analyzed using real-time RT-PCR.ResultsExpression of ASPN, TGF-β1, and TGF-β3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-β1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage.ConclusionsThe increased ratio of ASPN mRNA to TGF-β1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-β1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Combined microwave irradiation and intraarticular glutamine administration-induced HSP70 expression therapy prevents cartilage degradation in a rat osteoarthritis model.

The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real‐time RT‐PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression.

MDR1a/1b gene silencing enhances drug sensitivity in rat fibroblast-like synoviocytes.

Drug resistance mediated by P‐glycoprotein (P‐gp) is one of the major reasons for the failure of rheumatoid arthritis (RA) therapy with disease modifying anti‐rheumatic drugs and glucocorticoids. In the present study, we aimed to investigate the in vitro effectiveness of small interfering RNA (siRNA) to render rat fibroblast‐like synoviocytes (FLS) susceptible to drugs. We also attempted the electroporation‐mediated transfer of siRNA against multidrug resistance (MDR) genes into rat knee joints.

Lansoprazole Inhibits Nitric Oxide and Prostaglandin E 2 Production in Murine Macrophage RAW 264.7 Cells

Aberrantly activated macrophages, which overproduce inflammatory mediators, are involved in the pathogenesis of many inflammatory diseases. We analyzed the anti-inflammatory activity of lansoprazole (LPZ), a typical proton pump (P-ATPase) inhibitor, on RAW264.7 murine macrophages. Treatment of lipopolysaccharide (LPS)-stimulated RAW264.7 cells with LPZ inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2). Since P-ATPase expression was not observed in RAW264.7 cells, the anti-inflammatory effect of LPZ was independent of ATPase. In contrast, diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, decreased NO but not PGE2 levels. LPZ suppressed the LPS-stimulated production by RAW264.7 cells of reactive oxygen species (ROS), which plays an important role in inflammatory responses. ROS elevation in these cells was associated with NO but not PGE2 production, suggesting that LPZ inhibits NO production by suppressing NADPH oxidase activity. These findings suggest that LPZ may be useful in the treatment of many inflammatory diseases associated with activated macrophages.

Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte

The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real‐time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage.

164 HYALURONAN AND INTERMITTENT HYDROSTATIC PRESSURE SYNERGISTICALLY SUPPRESSED MMP-13 AND IL-6 EXPRESSIONS IN OSTEOBLASTS FROM OA SUBCHONDRAL BONE

Purpose: Various inflammatory cytokines and proteases are involved in the initiation and progression of osteoarthritis (OA). We previously reported that the expressions of matrix metalloproteinase-13 (MMP-13) and interleukin-6 (IL-6) were augumented in OA subchondral bone. The purpose of this study was to investigate the effects of hyaluronan (HA) and mechanical stress on osteoblasts isolated from OA subchondral bone. Methods: OA subchondral bone from the distal end of the femur was harvested from 9 patients at total knee arthroplasty. The subchondral bone underlying the degenerated articular cartilage was cut into small pieces and incubated in DMEM for 3 weeks, and then osteoblasts were isolated. Subchondral bone osteoblasts (SBOs) were cultured with DMEM conraining 30% fluorescent labeled HA for 48 hours and washed twice with PBS. The monolayer cultured cells were observed with a fluorescence microscope. As control, SBOs cultured without fluorescent labeled HA was used. SBOs were divided into 4 experimental groups. Control group: cultured without stimulation, HA group: incubated with HA (1000 mg/ml, 48 hours), IHP group: applied intermittent hydrostatic pressure (IHP) (1/2 Hz, 5 MPa, 60 minutes), and HA+IHP group: incubated with HA followed by IHP. Total RNA were extracted and mRNA expression was examined by real-time RT-PCR for MMP-13 and IL-6. In control group and HA+IHP group, culture supernatant was harvested 24 hours after the application of HA + IHP, and concentrations of IL-6 and MMP-13 were measured using an enzyme-linked immunosorbent assay (ELISA). Values were analyzed statistically by Tukey-kramer’s test and paired t-test and a p value less than 0.05 was considered significant. Results: In the fluorescent labeled HA group, fluorescence was observed in the area of cytoplasm but not in nuclei 48 hours after the administration. The mRNA expressions of MMP-13 of the each group compared to the control group were 101±18.2%, 89.3±14.1% and 51.2±7.5% respectively, indicating that MMP-13 expression in the HA + IHP group significantly decreased compared to those in the control group and in the HA group. The IL-6 mRNA of the each group were 76.6±11.9 %, 73.2±10.5% and 54.0±18.3%, indicating that IHP treatment and HA + IHP treatment significantly suppressed the IL-6 mRNA.The production of MMP-13 and IL-6 were 51.3±11.6 (pg/ml) and 70.8±25.6 in the control group. In the HA + IHP group, they were significantly reduced to 53.9±25.6 and 45.2±11.1. Conclusions: The role of subchondral bone attracts attention in the onset and/or progression of OA. It was reported that HA influences metabolism in subchondral bone, and that subchondral bone becomes more compliant and thereby reduces cartilage stress. However, the mechanism of the influence of HA on subchondral bone remains unclear. In this study, HA was exposed to osteoblasts, and the enhanced expressions of MMP-13 and IL-6 in OA osteoblasts were significantly suppressed by HA in combination with IHP. In the natural course of OA, a resorption of subchondral bone was documented in the early stage of OA and was considered to take an important role in the progression of the disease. In OA, neovascularization between cartilage and subchondral bone is observed while tidemark disappears. Therefore, intra-articular injected HA could reach the subchondral tissue. The results in this study suggest that intra-articular injection of HA in combination with appropriate exercise could suppress MMP-13 and IL-6 expressions in subchondral bone, which may prevent abnormal metabolism in osseous tissue in OA.

428 ANALYSIS OF ANTI-INFLAMMATORY EFFECTS OF LANSOPRAZOLE ON MACROPHAGES

Conclusions: Our working group has focused on the development of immune markers which useful in the assessment of OA. Pathogenic NA belongs mainly to the IgG isotype. IgG to inflammatory pain agents would be potent diagnostic markers for OA and may play pathogenic roles in the development of OA. Taken together (BK-IgG, DA-IgG, HA-IgG, PGE2-IgG), our results cold promote the understanding of the role of immunity in OA.