Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress.

OBJECTIVE: It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. DESIGN: Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. RESULTS: COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. CONCLUSION: Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

Oxidized LDL binding to LOX-1 enhances MCP-1 expression in cultured human articular chondrocytes.

OBJECTIVE: It has been suggested that oxidized low-density lipoprotein (ox-LDL) has some roles in progression of osteoarthritis. The purpose of this study is to investigate whether ox-LDL binding to lectin-like ox-LDL receptor 1 (LOX-1) enhances monocyte chemoattractant protein 1 (MCP-1) expression in cultured human articular chondrocytes (HACs). METHOD: The time course and dose response of MCP-1 mRNA expression and MCP-1 protein release into medium following ox-LDL stimulation were investigated using quantitative Real time PCR (delta-delta Ct method) and enzyme-linked immunosorbent assay (ELISA), respectively. To examine the receptor specificity of ox-LDL action, HACs were preincubated with anti-human LOX-1 monoclonal antibody (TS92). RESULTS: A time-course study revealed that MCP-1 mRNA expression increased 5.09+/-0.86 fold 12h after ox-LDL stimulation compared to time-0. ox-LDL stimulation increased MCP-1 protein level in conditioned medium in a time-dependent manner. Increased MCP-1 level was evident 6h after stimulation, reaching 830+/-91 pg/ml at 24h (33+/-8 pg/ml at time-0). Dose responses of MCP-1 expression were also evident in mRNA and protein levels. Pretreatment with TS92 markedly suppressed these stimulating effects of ox-LDL, although that with non-specific IgG did not. Native LDL did not affect MCP-1 expression. CONCLUSION: Our results suggest that ox-LDL enhances MCP-1 expression in HACs and supports the hypothesis that ox-LDL is involved in cartilage degeneration.

An unusual dorsal fracture-dislocation of the proximal interphalangeal joint.

A case of dorsal fracture-dislocation of the proximal interphalangeal joint in which the volar plate had been pulled from its distal attachment without bony or cartilaginous attachments and the lip of the volar plate had also been detached separately and was obstructing full flexion of the joint is presented. A tentative mechanism of causation of this unusual variant of a common injury is suggested.

Possible involvement of the oxidized low-density lipoprotein/lectin-like oxidized low-density lipoprotein receptor-1 system in pathogenesis and progression of human osteoarthritis.

OBJECTIVE: Using human cartilage samples and cultured chondrocytes, to assess the possible involvement of oxidized low-density lipoprotein (ox-LDL) and lectin-like ox-LDL receptor-1 (LOX-1) in pathogenesis and progression of osteoarthritis (OA). METHODS: Thirty-two cartilage samples were obtained from 16 patients with knee OA, and 12 Control samples from six with femoral neck fracture. LOX-1 mRNA expressions in 12 OA and six Control samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry for ox-LDL and LOX-1 was performed in all samples. The histological OA grade was assessed with the modified Mankin score. The relative percentage of the ox-LDL and LOX-1 immunopositive chondrocytes was calculated in all samples. The effects of ox-LDL on cell viability in cultured human chondrocytes were investigated by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and on proteoglycan synthesis by monitoring [35S] sulfate incorporation. RESULTS: There was a statistically significant difference between mean LOX-1/GAPDH (LOX-1/human glyceraldehyde-3-phosphate dehydrogenase) ratio of OA samples and that of Control samples (40.6%+/-10.3 and 11.9%+/-2.8, respectively, P<0.0001). The mean percentage of ox-LDL-positive cells was 23.0+/-15.7% in OA and 4.3+/-3.7% in Control cells (P=0.0002). The mean percentage of LOX-1-positive cells was 51.7+/-29.5% in OA and 10.0+/-8.1% in Control cells (P<0.0001). Both the ox-LDL immunoreactivity and the LOX-1 immunoreactivity were significantly correlated with the modified Mankin scores (R2=0.67 and 0.48, respectively; P<0.0001 for each). ox-LDL significantly reduced the human chondrocyte viability and proteoglycan synthesis, and pretreatment with anti-human LOX-1 monoclonal antibody reversed these effects. CONCLUSION: The ox-LDL/LOX-1 system may be involved in human OA.

Hypoxia-induced hyaluronan synthesis by articular chondrocytes: the role of nitric oxide.

OBJECTIVE: Articular cartilage is an avascular tissue in which chondrocytes are exposed to hypoxic conditions. We previously demonstrated that reactive oxygen species (ROS) induced apoptosis of chondrocytes. We also demonstrated that nitric oxide (NO) was induced when chondrocytes were exposed to hypoxia and that NO inhibited the ROS-induced apoptosis. Hyaluronan (HA) is a high molecular weight glycosaminoglycan whose antioxidative effects have been reported. The purpose of the present study was to determine whether HA synthesis was induced in chondrocytes exposed to hypoxia, and, if so, whether the hypoxia-induced HA synthesis is regulated by NO. METHODS: Bovine articular chondrocytes were used in this study. Levels of HA were determined by the sandwich enzyme-binding assay. Expression of HA synthase (HAS) was determined with reverse transcription-polymerase chain reaction. The production of NO was examined using the Griess reaction. We also determined inducible nitric oxide synthase (iNOS) enzyme synthesis using the histochemistry and Western blot analysis. RESULTS: Chondrocytes cultured under hypoxic conditions exhibited enhanced HA synthesis. When the NO inhibitors, L-NMMA and L-NAME, were added, the hypoxia-enhanced HA levels in the culture medium were significantly inhibited. CONCLUSIONS: Endogenous NO synthesis plays an important role in hypoxia-enhanced HA synthesis.

Cyclic tensile stretch stimulates the release of reactive oxygen species from osteoblast-like cells.

It is known that the excessive generation of reactive oxygen species (ROS) is a significant factor in tissue injury observed in many disease states. To determine whether extreme levels of mechanical stress applied to osteoblasts enhances ROS synthesis, we loaded cyclic tensile stretch on osteoblast-like HT-3 cells. Cyclic tensile stretch loaded on these cells clearly enhanced ROS synthesis in a time- and magnitude-dependent fashion. Cyclic tensile stretch also enhanced superoxide dismutase (SOD) activity. The disruption of microfilaments with cytochalasin D abolished the stress-induced ROS synthesis. Rotenone, an inhibitor of the mitochondrial electron transport chain, enhanced stress-induced ROS synthesis. These data suggest that actin filament and mitochondria are involved in this action.

Hypoxia-induced nitric oxide protects chondrocytes from damage by hydrogen peroxide.

OBJECTIVE: Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O2) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damage METHODS: Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [(35)S]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR. RESULTS: Cells maintained at 5% O2 had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage. CONCLUSIONS: Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.

Enhancement of nitric oxide and proteoglycan synthesis due to cyclic tensile strain loaded on chondrocytes attached to fibronectin.

OBJECTIVE: Mechanical stress is an essential factor in the pathogenesis of osteoarthrosis. We sought to determine whether the strain-mediated alteration in proteoglycan (PG) synthesis was modulated by nitric oxide (NO) synthesis. METHODS: Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [35S] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used. RESULTS: Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced. CONCLUSIONS: Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis.

Cyclic tensile stretch loaded on bovine chondrocytes causes depolymerization of hyaluronan: involvement of reactive oxygen species.

OBJECTIVE: We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. Decreased size of hyaluronan (HA), the major macromolecule in synovial fluid, to which it imparts viscosity, is reported in patients with arthritis. The purpose of this study was to determine the alteration in the molecular weight range of HA as a result of mechanical deformation loaded on the chondrocytes, as well as the involvement of ROS in this action. METHODS: ROS were generated via the oxidation of hypoxanthine by xanthine oxidase. Cyclic tensile stretch was loaded using a vacuum-operated instrument. Levels of HA were measured using a sandwich enzyme-binding assay. Superoxide dismutase (SOD) activity and ROS were measured using water-soluble tetrazolium and a chemiluminescent probe, respectively. RESULTS: ROS depolymerized HA molecules. Cyclic tensile stretch depolymerized HA and induced ROS. SOD inhibited not only ROS induction but also HA depolymerization caused by the mechanical stress. CONCLUSION: ROS play an important role in mechanical stress-induced HA depolymerization.

Hydrogen peroxide induces apoptosis of osteocytes: involvement of calcium ion and caspase activity.

We hypothesized that reactive oxygen species play an important role in avascular/ischemic osteonecrosis. When isolated chick osteocytes were cultured with hydrogen peroxide, annexin V binding, which is the earliest marker of apoptosis, increased in a dose-dependent fashion. Hydrogen peroxide also induced the activation of caspase-3 and increase in cytosolic Ca2+. Treatment with BAPTA/AM (cheletor of cytosolic Ca2+) and Ac-DEVD-cho (caspase inhibitor) attenuated hydrogen peroxide-induced apoptosis. These data demonstrated the signal transduction pathways that participate in this hydrogen peroxide-induced cell damage.

A case of systemic lupus erythematosus associated with trigger wrist.

Abstract A 54-year-old woman complained of a painful, swollen, and clicking left wrist for 1 year. She had an 8-year history of systemic lupus erythematosus (SLE), treated with oral prednisolone. Flexion and extension of all fingers were difficult to initiate on wrist extension, and movement was accompanied by a palpable click at the wrist. Magnetic resonance imaging (MRI) and tenography revealed the expanded sheath of the flexor tendons and well-defined round free bodies known as rice bodies. Synovectomy and excision of rice bodies resulted in complete disappearance of swelling and triggering of the wrist.

Comparison of the inhibitory effects of two types (90 kDa and 190 kDa) of hyaluronic acid on the expression of fibrinolytic factors in human synovial fibroblasts.

Abstract Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA.

Cyclic tensile stretch inhibition of nitric oxide release from osteoblast-like cells is both G protein and actin-dependent.

Recent reports indicate the alteration of nitric oxide (NO) synthesis with mechanical stress loaded on the osteoblast and NO is considered to have a significant role in mechanotransduction. We found the involvement of guanine-nucleotide-binding regulatory proteins (G proteins), especially Gi, in stress-inhibited NO release of osteoblast-like cells (JOR:17;593-597, 1999). To determine further the mechanism involved in this process, we measured c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activity under cyclic tensile stretch loaded on osteoblast-like cells. Cyclic stretch significantly enhanced JNK/SAPK activity and pertussis toxin clearly reversed stress-enhanced JNK/SAPK activity. Cytochalasin D, actin microfilament disrupting reagent, also abolished the stress activation of JNK/SAPK. We propose a model for signaling events induced by cyclic tensile stretch, namely a transmembrane mechanosensor which couples Gi-protein, actin cytoskeleton and finally activates JNK/SAPK activity of osteoblasts.

Interleukin-4 reversed the Interleukin-1-inhibited proteoglycan synthesis through the inhibition of NO release: a possible involvement of intracellular calcium ion.

Interleukin-1 (IL-1) causes cartilage degradation through nitric oxide (NO) synthesis. Although Interleukin-4 (IL-4) antagonizes the IL-1-mediated cartilage degradation, the precise mechanisms are not clear. We examined the effect of IL-4 on NO synthesis in parallel with intracellular Ca levels ([Ca(2+)]i) and proteoglycan (PG) synthesis. IL-4-inhibited IL-1-enhanced NO release in a dose-dependent manner. IL-1-enhanced [Ca(2+)]i in the chondrocytes, and IL-4 attenuated this increase. IL-4 reversed IL-1-inhibited PG synthesis. Accordingly, IL-4 reversed the IL-1-inhibited PG synthesis through the inhibition of NO release. An increase in [Ca(2+)]i with IL-1 is possibly involved in this action.

Hyaluronic acid inhibits the expression of u-PA, PAI-1, and u-PAR in human synovial fibroblasts of osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Intraarticular administration of hyaluronic acid (HA) has been widely used for the treatment of osteoarthritis (OA). Fibrinolysis is closely related to the pericellular proteolysis involved in inflammation. However, the role of HA in the regulation of fibrinolytic factors is not yet known. We investigated the effect of HA on the pericellular fibrinolytic system of human synovial fibroblasts derived from OA and rheumatoid arthritis (RA). METHODS: Human synovial fibroblasts obtained from OA and RA were cultured in the presence and absence of HA. The antigen of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured by ELISA, and u-PA activity was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunohistochemical analysis of u-PA were employed to detect u-PA receptor (u-PAR). RESULTS: HA suppressed the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium. Suppression of u-PA activity in OA synovial fibroblasts was more marked than in those of RA. The u-PA binding assay of OA and RA synovial fibroblasts revealed a single class of binding site: dissociation constant (Kd) 23.7 nM, maximal number of binding sites (Bmax) 3.11x10(4) binding sites/cell; Kd 16.5 nM, Bmax of 9.88x10(4) binding sites/cell, respectively. HA decreased Bmax in fibroblasts of both OA and RA. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease of the staining of u-PAR was more pronounced in the cells of RA than in OA. CONCLUSION: Pericellular fibrinolytic activity mediated by the u-PA/u-PAR system and PAI-1 was attenuated by HA in synovial fibroblasts derived from OA and RA. Thus, HA may be a useful agent to inhibit the inflammation of arthritis.

Effect of hydrogen peroxide on the metabolism of articular chondrocytes.

OBJECTIVE: To examine the effect of hydrogen peroxide on chondrocyte metabolism. MATERIALS AND METHODS: Bovine articular chondrocytes were used. Proteoglycan (PG) synthesis was measured with [35S] sulfate incorporation. For detection of apoptosis, the TdT-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V assay were used. Extracellular-regulated protein kinase (ERK) activity was measured using a mitogen-activated protein kinase assay system. RESULTS: Addition of hydrogen peroxide resulted in the inhibition of PG synthesis, apoptosis, and enhanced ERK activity. CONCLUSION: Hydrogen peroxide plays an important role in regulating the metabolism of chondrocytes.

Pertussis toxin-sensitive G proteins as mediators of stretch-induced decrease in nitric-oxide release of osteoblast-like cells.

Mechanical loading plays an important role in regulating bone remodeling, and nitric oxide may be one regulator of this process. To determine how mechanical stress modulates osteoblast function, we loaded cyclic tensile stretch on osteoblast-like cells and measured levels of nitric oxide in the medium. High frequency of stretch at any magnitude inhibited release of nitric oxide; however, low frequency of stretch enhanced its release from the static control. To examine the involvement of G protein (guanine nucleotide-binding regulatory protein) in stress-inhibited release of nitric oxide, we added pertussis toxin, a specific inhibitor of the Gi class, and found that it completely reversed the stress-inhibited release. These data support the idea that pertussis toxin-sensitive G protein is activated in the presence of cyclic tensile stretch.

Healing of segmental bone defects in rats induced by a beta-TCP-MCPM cement combined with rhBMP-2.

A beta-tricalcium phosphate-monocalcium phosphate monohydrate (beta-TCP-MCPM) cement was evaluated as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat femoral critical-size defects. Hard cement cylinders (4 x 5 mm) impregnated with two different doses of rhBMP-2 (1.26 or 6.28 microg) were implanted into each defect, and the results were compared with those in rats that had implantations of cylinders only. Implantation of the 6.28 microg dose of rhBMP-2 caused a large bone shell to form around the defect, resulting in osseous union in all cases within 3 weeks. Except for beta-TCP granules, the cement was resorbed and replaced by bone tissue at 6 weeks. A torsion test at 9 weeks showed that the failure torque and bone stiffness had recovered 99% and 141%, respectively, compared with the intact contralateral femur. The defects that received 1.26 microg of rhBMP-2 resulted in 40% union and 41% of the failure torque at 9 weeks. However, no instances of union were observed in the defects implanted with cylinders only. In conclusion, the beta-TCP-MCPM cement was shown to be effective as a rhBMP-2 carrier. Combined with rhBMP-2, this cement was rapidly resorbed and completely healed the defects.

Factors related to degradation of articular cartilage in osteoarthritis: a review.

OBJECTIVES: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. MATERIALS AND METHODS: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. RESULTS: Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. CONCLUSION: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.