Kuniaki Honjo

N-acetylcysteine prevents nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis.

We investigated whether N‐acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)‐induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0–2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase‐3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO‐induced apoptosis, ROS overproduction, p53 up‐regulation, and caspase‐3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis‐preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO‐induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.

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.

Silencing the expression of connexin 43 decreases inflammation and joint destruction in experimental arthritis.

The objective of the present study was to determine whether the expression of connexin 43 (Cx43) effected on inflammatory conditions in rat fibroblast‐like synoviocytes (FLS) and on rat model of rheumatoid arthritis (RA). The expression of Cx43 in rat FLS stimulated with lipopolysaccharide (LPS) was confirmed by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR). The effects of small‐interfering RNA targeting Cx43 (siCx43) on pro‐inflammatory cytokines and chemokine were assessed by real‐time RT‐PCR and enzyme‐linked immunosorbent assay (ELISA). The therapeutic and side effects of siCx43 in a rat model of collagen‐induced arthritis (CIA) were examined by in vivo electroporation method. LPS markedly enhanced Cx43 gene expression in rat FLS, with transfection of siCx43 suppressing the over‐expression of pro‐inflammatory cytokines and the chemokine. Treatment of CIA rats with siCx43 significantly ameliorated paw swelling, and significantly reduced histological arthritis scores and radiographic scores. In histological appearance of rat ankle joints, siCx43 treatment significantly decreased the number of tartrate‐resistant acid phosphatase (TRAP)‐positive (osteoclast‐like) cells. These findings indicated that siCx43 had anti‐inflammatory effects in rat FLS and efficiently inhibited the development of CIA. Cx43 may play an important role in the pathophysiology of RA, and may be a potential target molecule for novel RA therapies.

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.

HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF‐1α)‐dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2, which induces HIF‐1α, to simulate hypoxia, or transfected with siRNAs targeting HIF‐1α (si‐HIF‐1α) and HSP70 (si‐HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF‐1α under hypoxia or simulated hypoxia, but not in the presence of si‐HIF‐1α. Hypoxia‐induced overexpression of ECM genes was significantly suppressed by si‐HIF‐1α or si‐HSP70. Cell viability positively correlated with hypoxia, but transfection with si‐HIF‐1α or si‐HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside‐treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si‐HIF‐1α or si‐HSP70 almost completely blocked these effects. These findings indicated that HIF‐1α‐induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF‐1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression.

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.

Influence of extracellular matrix on the expression of inflammatory cytokines, proteases, and apoptosis-related genes induced by hydrostatic pressure in three-dimensionally cultured chondrocytes

BackgroundThe purpose of this study was to investigate the influence of hydrostatic pressure (HP) on the gene expression of cartilage matrix, cytokines, and apoptosis-associated factors in chondrocytes in which the cartilage was in extracellular matrix (ECM)-rich or ECM-poor condition.MethodsChondrocytes were isolated from rabbit joints and cultured in alginate beads. Immediately after embedding (0W group) or after 2 weeks culture (2W group), the amounts of glycosaminoglycan (GAG) in the alginate beads were quantified. Both groups were exposed to continuous HP of 10 or 50 MPa for 12 h. The expression of inflammatory cytokines, proteases, and apoptosis-related factors were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of proteoglycan core protein (PG) and collagen type II were quantified by real-time RT-PCR.ResultsAll of the GAG components in alginate beads markedly increased in the 2W group. The expression of PG and collagen type II increased after exposure to 10 MPa in both groups. In the 0W group, these levels decreased after exposure to 50 MPa of HP. The expression of interleukins IL-6 and IL-8 increased after exposure to HP in the 0W group. HP at 50 MPa induced mRNA expression of ADAMTS-5 in the 0W group but not in the 2W group. The expression of Fas increased after exposure to HP in the 0W group.ConclusionsThese findings suggested that nonphysiological, excessive HP on chondrocytes with the ECM in poor condition reduced matrix gene expression and increased expression of the genes associated with apoptosis and catabolism of the cartilage matrix. These results might therefore be associated with the pathogenesis of osteoarthritis.

Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

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.