Accelerated, aging-dependent development of osteoarthritis in alpha1 integrin-deficient mice

OBJECTIVE: Cell-matrix interactions regulate chondrocyte differentiation and survival. The alpha1beta1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin alpha1 gene (alpha1-KO mice) provide a model that can be used to address the role of cell-matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis. METHODS: Knee joints from alpha1-KO and wild-type (WT) BALB/c mice were harvested at ages 4-15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of alpha1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis. RESULTS: In WT mice, the alpha1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in alpha1-positive chondrocytes in the superficial and upper mid-zones in OA-affected areas in joints from old WT mice. The alpha1-KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP-2 and MMP-3 expression was increased in the OA-affected areas. In cartilage from alpha1-KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the alpha1 integrin subunit is involved in the early remodeling process in OA cartilage. CONCLUSION: Deficiency in the alpha1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging-dependent development of OA.     

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice

Objective

Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR‐1) in the development of osteoarthritis (OA) and its underlying mechanisms.

Methods

FGFR‐1 was deleted from the articular chondrocytes of adult mice in a cartilage‐specific and tamoxifen‐inducible manner. Two OA models (aging‐associated spontaneous OA, and destabilization‐induced OA), as well as an antigen‐induced arthritis (AIA) model, were established and tested in Fgfr1‐deficient and wild‐type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR‐1 inhibitor on human articular chondrocytes was examined.

Results

The gross morphologic features of Fgfr1‐deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12‐month‐old Fgfr1‐deficient mice displayed greater aggrecan staining compared to 12‐month‐old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR‐1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP‐13) and up‐regulation of FGFR‐3 in mouse and human articular chondrocytes.

Conclusion

Disruption of FGFR‐1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down‐regulation of MMP‐13 expression and up‐regulation of FGFR‐3 levels may contribute to the phenotypic changes observed in Fgfr1‐deficient mice.

     

Cilostazol protects rat chondrocytes against nitric oxide-induced apoptosis in vitro and prevents cartilage destruction in a rat model of osteoarthritis

OBJECTIVE: To examine whether cilostazol, a selective phosphodiesterase type III inhibitor, protects rat articular chondrocytes against nitric oxide (NO)-induced apoptosis and prevents cartilage destruction in mono-iodoacetate-induced osteoarthritis (OA) in a rat model in which inducible nitric oxide synthase (iNOS) is expressed. METHODS: The NO donor sodium nitroprusside was administered to rat articular chondrocytes that had been pretreated with cilostazol. Induction of apoptosis was evaluated by DNA electrophoresis and pulsed-field gel electrophoresis. The expression level and the subcellular location of apoptosis-associated factors were examined by Western blot analysis and confocal microscopy, respectively. Protein kinase CK2 (PKCK2) activity was also assayed. To examine whether orally administered cilostazol prevents cartilage destruction in vivo, cartilage samples obtained from rats with experimentally induced OA were subjected to hematoxylin and eosin, Safranin O, and TUNEL staining and immunohistochemical analysis of iNOS expression. RESULTS: Cilostazol prevented NO-induced reduction in viability, in a dose-dependent manner. It also prevented the up-regulation of phosphorylated p53 and p38, the down-regulation of heme oxygenase 1, the subcellular translocation of apoptosis-inducing factor and cytochrome c, and the activation of caspases 3, 7, and 8 induced by NO treatment, indicating that cilostazol prevented NO-induced cell death by blocking apoptosis. In addition, cilostazol prevented NO-induced translocation of cleaved Bid onto mitochondria, and caused phosphorylated Bid to accumulate in the nucleus and cytosol. Cilostazol prevented the down-regulation of PKCK2 and the reduction in PKCK2 activity induced by NO, indicating that its apoptosis-preventing activity was mediated via PKCK2. It also prevented chondrocyte apoptosis and cartilage destruction in a rat model of experimentally induced OA. CONCLUSION: Our findings indicate that cilostazol prevents NO-induced apoptosis of chondrocytes via PKCK2 in vitro and prevents cartilage destruction in a rat model of OA.     

Increased expression of the collagen receptor discoidin domain receptor 2 in articular cartilage as a key event in the pathogenesis of osteoarthritis

OBJECTIVE: To investigate the role of the collagen receptor discoidin domain receptor 2 (DDR-2) in the pathogenesis of osteoarthritis (OA). METHODS: Histologic and immunohistochemical analyses were performed to characterize femoral head cartilage from 7 patients with OA and 4 patients with fracture, as well as articular cartilage from the knee joints of mice with surgically induced OA. Gene constructs encoding human Raf kinase inhibitor protein (RKIP), DDR-2 lacking the discoidin (DS) domain (DeltaDS-DDR-2) or the protein tyrosine kinase (PTK) core (DeltaPTK-DDR-2), DDR-2 containing a substitution of tyrosine for alanine at position 740 (Y740A), and luciferase driven by the matrix metalloproteinase 13 (MMP-13) promoter were transfected into human chondrocyte cell lines. Activated and neutralized alpha2beta1 integrin polyclonal antibodies, interleukin-1 receptor antagonist, and the chemical inhibitors SB203580, for p38, and SP600125, for JNKs, were used in cell cultures. Real-time polymerase chain reaction was performed to examine MMP-13 and DDR-2 messenger RNA (mRNA). RESULTS: Increased immunostaining for DDR-2, MMP-13, and MMP-derived type II collagen fragments was detected in cartilage from patients with OA and from mice with surgically induced OA. The discoidin domain and PTK core of DDR-2 were essential for signal transmission and the resulting increased expression of MMP-13 in chondrocytes. Y740A mutation of DDR-2 reduced levels of mRNA for MMP-13 and endogenous DDR-2. The overexpression of RKIP or preincubation with the p38 inhibitor reduced MMP-13 mRNA levels. DDR-2 signaling was independent of the alpha2beta1 integrin and the interleukin-1-induced signaling pathways in chondrocytes. CONCLUSION: These findings suggest that increased expression of DDR-2, resulting in the elevated expression of MMP-13, may be one of the common events in OA progression.     

Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice

Rheumatoid arthritis is one of the most critical diseases that impair the quality of life of patients, but its pathogenesis has not yet been fully understood. Osteopontin (OPN) is an extracellular matrix protein containing Arg-Gly-Asp (RGD) sequence, which interacts with alpha(v)beta3 integrins, promotes cell attachment, and cell migration and is expressed in both synovial cells and chondrocytes in rheumatoid arthritis; however, its functional relationship to arthritis has not been known. Therefore, we investigated the roles of OPN in the pathogenesis of inflammatory process in a rheumatoid arthritis model induced by a mixture of anti-type II collagen mAbs and lipopolysaccharide (mAbs/LPS). mAbs/LPS injection induced OPN expression in synovia as well as cartilage, and this expression was associated with joint swelling, destruction of the surface structures of the joint based on scanning electron microscopy, and loss of toluidine blue-positive proteoglycan content in the articular cartilage in wild-type mice. In contrast, OPN deficiency prevented the mice from such surface destruction, loss of proteoglycan in the articular joint cartilage, and swelling of the joints even when the mice were subjected to mAbs/LPS injection. Furthermore, mAbs/LPS injection in wild-type mice enhanced the levels of CD31-positive vessels in synovia and terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive chondrocytes in the articular cartilage, whereas such angiogenesis as well as chondrocyte apoptosis was suppressed significantly in OPN-deficient mice. These results indicated that OPN plays a critical role in the destruction of joint cartilage in the rheumatoid arthritis model in mice via promotion of angiogenesis and induction of chondrocyte apoptosis.     

Statin prevents chondrocyte aging and degeneration of articular cartilage in osteoarthritis (OA)

Recent reports have shown that statin (HMG-CoA reductase inhibitors) may have the potential to inhibit inflammatory arthritis. More recently, the idea that chondrocyte aging is closely associated with the progression of cartilage degeneration has been promulgated. Here, we demonstrate the potential of statin as protective agents against chondrocyte aging and degeneration of articular cartilage during the progression of osteoarthritis (OA), both in vitro and in vivo. The OA-related catabolic factor, IL-1β induced marked downregulation of cellular activity, expression of a senescent biomarker, specific senescence-associated β-galactosidase activity and shortening of the cellular lifespan in chondrocytes. In contrast, treatment with statin inhibited the IL-1β-induced production of cartilage matrix degrading .enzymes (metalloprotease-1 and -13) and cellular senescence in of chondrocytes in vitro. In addition, this statin accelerated the production of cartilage matrix proteoglycan in chondrocytes. The in vivo study was performed on the STR/OrtCrlj mouse, an experimental model which spontaneously develops an osteoarthritic process. In this mouse model, treatment with statin significantly reduced the degeneration of articular cartilage, while the control knee joints showed progressive cartilage degeneration over time. These findings suggest that statin may have the potential to prevent the catabolic stress-induced chondrocyte disability and aging observed in articular cartilage. Our results indicate that statin are potential therapeutic agents for protection of articular cartilage against the progression of OA.     

Accelerated,aging‐dependent development of osteoarthritis in α1 integrin–deficient mice

Objective

Cell–matrix interactions regulate chondrocyte differentiation and survival. The α1β1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin α1 gene (α1‐KO mice) provide a model that can be used to address the role of cell–matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis.

Methods

Knee joints from α1‐KO and wild‐type (WT) BALB/c mice were harvested at ages 4–15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of α1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis.

Results

In WT mice, the α1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in α1‐positive chondrocytes in the superficial and upper mid‐zones in OA‐affected areas in joints from old WT mice. The α1‐KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP‐2 and MMP‐3 expression was increased in the OA‐affected areas. In cartilage from α1‐KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the α1 integrin subunit is involved in the early remodeling process in OA cartilage.

Conclusion

Deficiency in the α1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging‐dependent development of OA.

     

Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis

OBJECTIVE: Chondromodulin-I (ChM-I), a cartilage derived anti-angiogenic factor, has been shown to regulate the vascular invasion during endochondral bone formation. We evaluated the expression and localization of ChM-I in articular cartilage during the progression of osteoarthritis (OA) in the rat, and correlated ChM-I expression with the increase in vascular invasion into OA articular cartilage. METHODS: Expression of ChM-I, type II collagen, basic fibroblast growth factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases MMP-9 and MMP-13 were examined in articular cartilage of intact growing and adult rats and in the surgically induced OA model using in situ hybridization, Western blot analysis, and immunohistochemistry. Co-immunostaining for ChM-I and CD-31 was performed to localize ChM-I and neovascularization in articular cartilage at advanced stage of OA. RESULTS: Abundant expression of ChM-I protein was detected in avascular regions of the developing and adult healthy articular cartilage. In early OA, ChM-I expression decreased in the superficial zone of articular cartilage, while levels of proteoglycan and type II collagen were comparable to control. In advanced OA, ChM-I expression was reduced in all zones of articular cartilage, and the number of VEGF-expressing cells was increased. Immunohistochemical studies showed that vascular invasion occurred in proximity to chondrocytes with high expression of pro-angiogenic markers, and decreased expression of ChM-I. CONCLUSION: High expression of ChM-I was detected in articular cartilage of growing and normal adult joints, implicating its role in the maintenance of avascularity of intact articular cartilage. Expression of ChM-I decreased, while expression of VEGF and other pro-angiogenic factors increased, in OA cartilage. These findings suggest the loss of ChM-I from articular cartilage might be responsible in part for promoting blood vessel invasion into the cartilage during progression of OA.     

Effect of NOS2 gene deficiency on the development of autoantibody mediated arthritis and subsequent articular cartilage degeneration

OBJECTIVE: To determine the effect of NOS2 gene deletion on articular cartilage degradation in autoantibody mediated arthritis (AMA). METHODS: Female C57BL/6Ai-[ko] NOS2 N5 (NOS2-/-) mice (7-8 weeks old) and the counterpart C57/Bl6 Crj mice (wild-type, WT) were studied. Arthritis was induced by intraperitoneal injection of 4 mg of an arthritogenic cocktail of 4 monoclonal antibodies raised against type II collagen twice on Day 0 and Day 1 followed by intraperitoneal injection of 50 micro g of lipopolysaccharide on Day 2. Individual limbs were scored for arthritis in 4 grades; the total maximum score per mouse was 16. Femoral condyles and tibial plateaus of both knee joints were collected on Day 15 for immunohistological studies on nitrotyrosine and matrix metalloproteinase (MMP)-3 and -9. DNA fragmentation in chondrocytes was detected by the nick-end labeling (TUNEL) method. Blood was also collected on Day 15 to determine serum levels of nitrite/nitrate and interleukin 1 beta (IL-1 beta). RESULTS: Both NOS2-/- and WT mice with AMA developed clinically apparent arthritis. In WT mice, the arthritis progressed rapidly and reached the peak score 11.4 +/- 2.9 on Day 12, whereas the arthritis in NOS2-/- mice was milder and the peak score was 7.7 +/- 2.8 on Day 13 (p < 0.05). The serum nitrite/nitrate levels, histological grades of articular cartilage degradation, and numbers of apoptotic chondrocytes and nitrotyrosine positive chondrocytes were significantly lower in NOS2-/- mice with AMA than in WT mice with AMA. Conversely, significant differences were not observed in MMP-3 or -9 expression in chondrocytes, or in serum IL-1 beta levels between these 2 groups of mice. CONCLUSION: NOS2 gene deletion did not affect the inflammatory responses, but reduced the cartilage degradation.     

Water-soluble C60 fullerene prevents degeneration of articular cartilage in osteoarthritis via down-regulation of chondrocyte catabolic activity and inhibition of cartilage degeneration during disease development

OBJECTIVE: Studies have shown the roles of oxidative stress in the pathogenesis of osteoarthritis (OA) and induction of chondrocyte senescence during OA progression. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against catabolic stress-induced degeneration of articular cartilage in OA, both in vitro and in vivo. METHODS: In the presence or absence of C60 (100 microM), human chondrocytes were incubated with interleukin-1beta (10 ng/ml) or H2O2 (100 microM), and chondrocyte activity was analyzed. An animal model of OA was produced in rabbits by resection of the medial meniscus and medial collateral ligament. Rabbits were divided into 5 subgroups: sham operation or treatment with C60 at 0.1 microM, 1 microM, 10 microM, or 40 microM. The left knee joint was injected intraarticularly with water-soluble C60 (2 ml), while, as a control, the right knee joint received 50% polyethylene glycol (2 ml), once weekly for 4 weeks or 8 weeks. Knee bone and cartilage tissue were prepared for histologic analysis. In addition, in the OA rabbit model, the effect of C60 (10 microM) on degeneration of articular cartilage was compared with that of sodium hyaluronate (HA) (5 mg/ml). RESULTS: C60 (100 microM) inhibited the catabolic stress-induced production of matrix-degrading enzymes (matrix metalloproteinases 1, 3, and 13), down-regulation of matrix production, and apoptosis and premature senescence in human chondrocytes in vitro. In rabbits with OA, treatment with water-soluble C60 significantly reduced articular cartilage degeneration, whereas control knee joints showed progression of cartilage degeneration with time. This inhibitory effect was dose dependent, and was superior to that of HA. Combined treatment with C60 and HA yielded a significant reduction in cartilage degeneration compared with either treatment alone. CONCLUSION: The results indicate that C60 fullerene is a potential therapeutic agent for the protection of articular cartilage against progression of OA.     

Regulation of tenascin-C expression by tumor necrosis factor-alpha in cultured human osteoarthritis chondrocytes

Expression of tenascin-C reappears in articular cartilage of persons with osteoarthritis (OA), while it is almost abolished in normal mature cartilage. Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is upregulated in OA cartilage and is involved in the progression of OA, and stimulates tenascin-C expression in other types of cells. We investigated regulation of tenascin-C expression by TNF-alpha through nuclear factor-alphaB (NF-kappaB) in OA cartilage in vivo and in vitro. METHODS: Human articular cartilages were obtained from patients with OA and immunofluorescence examination of tenascin-C and the activated RelA subunit was performed. Cultured chondrocytes isolated from human OA cartilage were treated with TNF-alpha and with SN50. Activation of RelA subunit of NF-kappaB was examined by immunolabeling. Changes in tenascin-C protein concentrations were determined by immunofluorescence of cells after monensin treatment and Western blot analysis of the cell lysates, and mRNA levels were analyzed by quantitative real-time polymerase chain reaction. RESULTS: Increased intensity of tenascin-C staining was observed in the damaged cartilage compared with normal cartilage. Activated RelA staining in chondrocyte nuclei was prominent in tenascin-C-positive areas of OA cartilage. Treatment of cultured chondrocytes by TNF-alpha induced translocation of activated RelA to the nuclei, followed by upregulation of tenascin-C expression in both mRNA and protein. Treatment with SN50 inhibited increases of RelA and tenascin-C expression in chondrocytes. CONCLUSION: TNF-alpha stimulated tenascin-C expression through NF-kappaB signaling with RelA activation in cultured OA chondrocytes, suggesting involvement of tenascin-C in OA cartilage remodeling.     

A type I collagen defect leads to rapidly progressive osteoarthritis in a mouse model

OBJECTIVE: This study was undertaken to test the hypothesis that abnormalities of the subchondral bone can result in osteoarthritis (OA). METHODS: We used a knockin model of human osteogenesis imperfecta, the Brittle IV (Brtl) mouse, in which defective type I collagen is expressed in bone. OA in individual mice was documented by micro-magnetic resonance imaging (micro-MRI) and micro-computed tomography (micro-CT). Alterations in the knee joints were confirmed by histopathologic and immunohistochemical analysis. In addition, atomic force microscopy (AFM) was used to assess the ultrastructure of the articular cartilage and subchondral bone matrix. RESULTS: Brtl mice had decreased integrity of bone but initially normal articular cartilage. However, by the second month of life, Brtl mice developed alterations of the cartilage that were characteristic of OA, as documented by micro-CT, micro-MRI, and histologic evaluation. In addition, chondrocyte loss and breakdown of the collagen matrix in the residual cartilage were demonstrated using AFM. CONCLUSION: The Brtl mouse model demonstrates that progressive destruction of articular cartilage characteristic of OA may be secondary to altered architecture of the underlying subchondral bone.     

Inhibition of beta-catenin signaling in articular chondrocytes results in articular cartilage destruction

OBJECTIVE: Osteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/beta-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of beta-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit beta-catenin signaling in chondrocytes. METHODS: The expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x(L), and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes. RESULTS: Expression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICAT-transgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x(L) expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice. CONCLUSION: Inhibition of beta-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICAT- transgenic mice.     

Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy.

OBJECTIVE: To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. METHODS. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. RESULTS. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. CONCLUSION. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.