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1.
Martine Roudier Xiaodong Li Qing‐Tian Niu Efrain Pacheco James K. Pretorius Kevin Graham Bo‐Rin P. Yoon Jianhua Gong Kelly Warmington Hua Z. Ke Roy A. Black Joanne Hulme Philip Babij 《Arthritis \u0026amp; Rheumatology》2013,65(3):721-731
Objective
Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage.Methods
Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin‐neutralizing monoclonal antibody (Scl‐Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl‐Ab or intraarticular (IA) delivery of a sclerostin antibody–Fab (Scl‐Fab) fragment.Results
Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin‐KO mice showed no difference in histopathologic features of the knee joint compared to age‐matched wild‐type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl‐Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl‐Ab or IA injection of Scl‐Fab had no effect on lesion development or severity.Conclusion
Genetic absence of sclerostin does not alter the normal development of age‐dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl‐Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.2.
3.
Martina Kaiser Jochen Haag Stephan Sder Brigitte Bau Thomas Aigner 《Arthritis \u0026amp; Rheumatology》2004,50(11):3535-3540
Objective
Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.Methods
RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.Results
Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.Conclusion
Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.4.
5.
Mark G. Chambers Lucy Cox Lisa Chong Nida Suri Patricia Cover Michael T. Bayliss Roger M. Mason 《Arthritis \u0026amp; Rheumatology》2001,44(6):1455-1465
Objective
To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice.Methods
The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry.Results
VDIPEN neoepitope was detected mainly in the pericellular matrix of deep‐zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper‐zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice.Conclusion
MMP‐ and aggrecanase‐generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.6.
7.
Atsushi Fukai Naohiro Kawamura Taku Saito Yasushi Oshima Toshiyuki Ikeda Fumitaka Kugimiya Akiro Higashikawa Fumiko Yano Naoshi Ogata Kozo Nakamura Ung‐Il Chung Hiroshi Kawaguchi 《Arthritis \u0026amp; Rheumatology》2010,62(3):826-836
Objective
To examine the role of the phosphoinositide‐dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification.Methods
Skeletal phenotypes of homozygous Akt1‐deficient (Akt1−/−) mice and their wild‐type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1.Results
Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1−/− chondrocytes, indicating a dominant role of Akt1. Akt1−/− mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild‐type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1−/− joints. Calcification was significantly suppressed in cultures of Akt1−/− chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1.Conclusion
Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA.8.
9.
Kazuo Yudoh Kiyoshi Shishido Hideki Murayama Mitsunobu Yano Kenji Matsubayashi Hiroya Takada Hiroshi Nakamura Kayo Masuko Tomohiro Kato Kusuki Nishioka 《Arthritis \u0026amp; Rheumatology》2007,56(10):3307-3318
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 μM), human chondrocytes were incubated with interleukin‐1β (10 ng/ml) or H2O2 (100 μM), 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 μM, 1 μM, 10 μM, or 40 μM. 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 μM) on degeneration of articular cartilage was compared with that of sodium hyaluronate (HA) (5 mg/ml).Results
C60 (100 μM) 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.10.
Lin Xu Jacqueline Servais Ilona Polur Doil Kim Peter L. Lee Kimberly Chung Yefu Li 《Arthritis \u0026amp; Rheumatology》2010,62(9):2736-2744
Objective
To investigate whether the reduction of discoidin domain receptor 2 (DDR‐2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).Methods
Double‐heterozygous (type XI collagen–deficient [Col11a1+/−] and Ddr2‐deficient [Ddr2+/−]) mutant mice were generated. Knee joints of Ddr2+/− mice were subjected to microsurgical destabilization of the medial meniscus. Conditions of the articular cartilage from the knee joints of the double‐heterozygous mutant and surgically treated mice were examined by histology, evaluated using a modified Mankin scoring system, and characterized by immunohistochemistry.Results
The rate of progressive degeneration in knee joints was dramatically reduced in the double‐heterozygous mutant mice compared with that in the type XI collagen–deficient mice. The progression in the double‐heterozygous mutant mice was delayed by ∼6 months. Following surgical destabilization of the medial meniscus, the progressive degeneration toward OA was dramatically delayed in the Ddr2+/− mice compared with that in their wild‐type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of DDR‐2 and matrix metalloproteinase 13.Conclusion
Reduction of DDR‐2 expression attenuates the articular cartilage degeneration of knee joints induced either by type XI collagen deficiency or by surgical destabilization of the medial meniscus.11.
12.
Merry Z. C. Ruan Brian Dawson Ming‐Ming Jiang Francis Gannon Michael Heggeness Brendan H. L. Lee 《Arthritis \u0026amp; Rheumatology》2013,65(2):388-396
Objective
The mouse is an optimal model organism in which gene–environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice is based on histopathology and immunohistochemistry, which are labor‐intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques.Methods
A novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high‐resolution micro–computed tomography (micro‐CT) and phase‐contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM).Results
Phase‐contrast micro‐CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase‐contrast micro‐CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase‐contrast micro‐CT.Conclusion
We established an imaging technology that comprehensively assessed and quantified the 2‐dimensional and 3‐dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high‐throughput assessment of genetic and therapeutic models of OA in mice.13.
14.
Sohel M. Julovi Tadashi Yasuda Makoto Shimizu Teruko Hiramitsu Takashi Nakamura 《Arthritis \u0026amp; Rheumatology》2004,50(2):516-525
Objective
To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.Methods
IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.Results
Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.Conclusion
This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.15.
L. Xu C. M. Flahiff B. A. Waldman D. Wu B. R. Olsen L. A. Setton Y. Li 《Arthritis \u0026amp; Rheumatology》2003,48(9):2509-2518
Objective
To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.Methods
Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.Results
The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.Conclusion
Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.16.
C. Grimmer D. Pfander B. Swoboda T. Aigner L. Mueller F. F. Hennig K. Gelse 《Arthritis \u0026amp; Rheumatology》2007,56(12):4084-4094
17.
Objective
Dkk is a family of canonical Wnt antagonists with 4 members (Dkk‐1, Dkk‐2, Dkk‐3, and Dkk‐4). We undertook this study to explore the roles of Dkk‐1 and Dkk‐2 in osteoarthritic (OA) cartilage destruction in mice.Methods
Expression of Dkk and other catabolic factors was determined at the messenger RNA and protein levels in human and mouse OA cartilage. Experimental OA in mice was induced by destabilization of the medial meniscus (DMM) or by intraarticular injection of Epas1 adenovirus (AdEPAS‐1). The role of Dkk in OA pathogenesis was examined by intraarticular injection of AdDkk‐1 or by using chondrocyte‐specific Dkk1 (Col2a1‐Dkk1)–transgenic mice and Dkk2 (Col2a1‐Dkk2)–transgenic mice. Primary culture mouse chondrocytes were also treated with recombinant Dkk proteins.Results
We found opposite patterns of Dkk1 and Dkk2 expression in human and mouse experimental OA cartilage: Dkk1 was up‐regulated and Dkk2 was down‐regulated. Overexpression of Dkk1 by intraarticular injection of AdDkk‐1 significantly inhibited DMM‐induced experimental OA. DMM‐induced OA was also significantly inhibited in Col2a1‐Dkk1–transgenic mice compared with their wild‐type littermates. However, Col2a1‐Dkk2–transgenic mice showed no significant difference in OA pathogenesis. Wnt‐3a, which activates the canonical Wnt pathway, induced Mmp13 and Adamts4 expression in primary culture chondrocytes, an effect that was significantly inhibited by Dkk‐1 pretreatment or Dkk1 overexpression.Conclusion
Our findings indicate that expression of Dkk1, but not Dkk2, in chondrocytes inhibits OA cartilage destruction. The protective effect of Dkk‐1 appears to be associated with its capacity to inhibit Wnt‐mediated expression of catabolic factors, such as Mmp13, providing evidence that Dkk‐1 might serve as a therapeutic target for OA treatment.18.
19.
Takumi Kakinuma Tadashi Yasuda Takefumi Nakagawa Teruko Hiramitsu Miki Akiyoshi Masao Akagi Tatsuya Sawamura Takashi Nakamura 《Arthritis \u0026amp; Rheumatology》2004,50(11):3495-3503
Objective
To investigate for the presence of oxidized low‐density lipoprotein (ox‐LDL) and lectin‐like oxidized LDL receptor 1 (LOX‐1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox‐LDL with LOX‐1 can induce matrix metalloproteinase 3 (MMP‐3) in articular cartilage explant culture.Methods
Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX‐1 and ox‐LDL by confocal fluorescence microscopy. The association between ox‐LDL and LOX‐1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox‐LDL, with or without preincubation with neutralizing anti–LOX‐1 antibody. MMP‐3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme‐linked immunosorbent assay.Results
The majority of the RA chondrocytes stained positively with both anti–LOX‐1 and anti–ox‐LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti–LOX‐1 antibody suppressed the binding of DiI‐labeled ox‐LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX‐1. In contrast to native LDL, ox‐LDL induced MMP‐3 synthesis by articular chondrocytes in association with the induction of LOX‐1, which resulted in enhanced secretion of MMP‐3 into the culture medium. Anti–LOX‐1 antibody reversed ox‐LDL–stimulated MMP‐3 synthesis to control levels.Conclusion
Ox‐LDL, principally mediated by LOX‐1, enhanced MMP‐3 production in articular chondrocytes. Increased accumulation of ox‐LDL with elevated expression of LOX‐1 in RA cartilage indicates a specific role of the receptor–ligand interaction in cartilage pathology in RA.20.
Beatriz Carams Noboru Taniguchi Shuhei Otsuki Francisco J. Blanco Martin Lotz 《Arthritis \u0026amp; Rheumatology》2010,62(3):791-801