A longitudinal analysis of urinary biochemical markers and bone mineral density in STR/Ort mice as a model of spontaneous osteoarthritis

Objective

To investigate the longitudinal changes both in the urinary concentrations of biochemical markers and in bone mineral density (BMD) during disease progression in the STR/Ort mouse model of osteoarthritis (OA).

Methods

Male STR/Ort mice were studied, with CBA mice used as nonarthritic controls. Radiographic evaluation and grading of the knee and measurements of urinary C‐terminal crosslinking telopeptide of type II collagen (CTX‐II), pyridinoline (Pyr), and deoxypyridinoline were performed between 8 weeks and 40 weeks of age. The BMD of the femoral shaft was measured from 20 weeks to 40 weeks of age and adjusted for body weight. Histologic evaluation and grading were performed at 40 weeks of age. STR/Ort mice were divided into 2 subgroups (STR OA and STR non‐OA) based on histologic grading.

Results

No significant differences between STR/Ort and CBA mice were observed for any biochemical marker or BMD at any time point. Urinary CTX‐II levels and BMD in the STR OA subgroup were higher than those in the STR non‐OA subgroup before radiographic changes of OA were apparent. Higher urinary Pyr levels in the STR OA subgroup were observed at the advanced stage of OA.

Conclusion

Urinary CTX‐II could be a useful marker in the early diagnosis and predicting the progression of OA, and urinary Pyr may be a potential marker to assess the severity of OA at an advanced stage. An increase in BMD prior to the establishment of radiographic OA may be related to the induction of OA.

     

Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor β–induced osteophytes: Limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation

Objective

Osteoarthritis (OA) is characterized by cartilage damage, synovial fibrosis, and osteophyte formation. Both transforming growth factor β (TGFβ) and bone morphogenetic protein 2 (BMP‐2) can induce the formation of osteophytes during OA, but their specific role in this process is unclear. The purpose of this study was to investigate the respective contributions of TGFβ and BMP‐2 to OA.

Methods

Mouse knee joints injected with adenovirus (Ad‐TGFβ or Ad‐BMP‐2) were compared histologically with knee joints from murine models of OA (joints injected with collagenase and joints from STR/Ort mice with spontaneous OA). To further investigate the role of BMP during osteophyte formation, adenovirus Ad‐Gremlin was injected into knee joints that had previously been injected with Ad‐TGFβ or collagenase.

Results

BMP‐2 induced early osteophytes, which bulged from the growth plates on the femur and grew on top of the patella, whereas TGFβ induced early osteophyte formation on the bone shaft beneath the collateral ligament on the femur as well as on top of the patella. The pattern of osteophyte formation during experimental OA closely resembled that of TGFβ‐induced osteophyte formation, but differed from the pattern induced by BMP‐2. Ad‐Gremlin proved to be able to totally block BMP‐2–induced osteophyte formation. However, blocking BMP activity inhibited neither TGFβ‐induced nor experimental OA–associated osteophyte formation.

Conclusion

Our findings demonstrate that the role of BMP during the onset of TGFβ‐induced and experimental OA–induced osteophyte formation is limited. The latter finding does not rule out a role of BMP during osteophyte maturation.

     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice

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.

     

In vivo bone‐specific EphB4 overexpression in mice protects both subchondral bone and cartilage during osteoarthritis

Objective

In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone‐specific EphB4 overexpression on OA pathophysiology.

Methods

Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10‐week‐old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10‐week‐old male EphB4 homozygous transgenic (EphB4‐Tg) and wild‐type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro–computed tomography.

Results

There was no obvious phenotype difference in skeletal development between EphB4‐Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post‐DMM, the EphB4‐Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4‐Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate‐resistant acid phosphatase–positive osteoclasts at both times assessed post‐DMM in the EphB4‐Tg mice than in the WT mice.

Conclusion

This is the first in vivo evidence that bone‐specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.

     

Inhibition of inflammatory bone erosion by constitutively active STAT‐6 through blockade of JNK and NF‐κB activation

Objective

NF‐κB and JNK signaling pathways play key roles in the pathogenesis of inflammatory arthritis. Both factors are also activated in response to osteoclastogenic factors, such as RANKL and tumor necrosis factor α. Inflammatory arthritis and bone erosion subside in the presence of antiinflammatory cytokines such as interleukin‐4 (IL‐4). We have previously shown that IL‐4 inhibits osteoclastogenesis in vitro through inhibition of NF‐κB and JNK activation in a STAT‐6–dependent manner. This study was undertaken to investigate the potential of constitutively active STAT‐6 to arrest the activation of NF‐κB and JNK and to subsequently ameliorate the bone erosion associated with inflammatory arthritis in mice.

Methods

Inflammatory arthritis was induced in wild‐type and STAT‐6–null mice by intraperitoneal injection of arthritis‐eliciting serum derived from K/B×N mice. Bone erosion was assessed in the joints by histologic and immunostaining techniques. Cell‐permeable Tat‐STAT‐6 fusion proteins were administered intraperitoneally. Cells were isolated from bone marrow and from joints for the JNK assay, the DNA‐binding assays (electrophoretic mobility shift assays), and for in vitro osteoclastogenesis.

Results

Activation of NF‐κB and JNK in vivo was increased in extracts of cells retrieved from the joints of arthritic mice. Cell‐permeable, constitutively active STAT‐6 (i.e., STAT‐6‐VT) was effective in blocking NF‐κB and JNK activation in RANKL‐treated osteoclast progenitors. More importantly, STAT‐6‐VT protein significantly inhibited the in vivo activation of NF‐κB and JNK, attenuated osteoclast recruitment in the inflamed joints, and decreased bone destruction.

Conclusion

Our findings indicate that the administration of STAT‐6‐VT presents a novel approach to the alleviation of bone erosion in inflammatory arthritis.

     

Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury

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.

     

Quality of well‐being in older people with osteoarthritis

Objective

To examine the sensitivity of the Quality of Well‐Being Scale (QWB) as a measure of health‐related quality of life (HRQOL) in people with osteoarthritis (OA).

Methods

The QWB was administered, along with the Arthritis Impact Measurement Scale (AIMS) and other health measures. Health care utilization data were also obtained.

Results

People with OA had a mean QWB score of 0.643. The QWB scores were significantly correlated with total AIMS scores, self‐rated health status, health care costs, depression scores, and most AIMS subscales. In addition, changes in QWB scores after 1 year were significantly correlated with changes in total AIMS scores and some AIMS subscales.

Conclusion

The QWB appears to be a useful and sensitive generic, utility‐based measure of HRQOL in people with OA.

     

A20 suppresses inflammatory responses and bone destruction in human fibroblast‐like synoviocytes and in mice with collagen‐induced arthritis

Objective

Nuclear factor‐κB (NF‐κB) has been implicated as a therapeutic target for the treatment of rheumatoid arthritis (RA). The purpose of this study was to determine whether A20, a universal inhibitor of NF‐κB, might have antiarthritic effects.

Methods

An adenovirus containing A20 complementary DNA (AdA20) was used to deliver A20 to human rheumatoid fibroblast‐like synoviocytes (FLS) in vitro as well as to mice with collagen‐induced arthritis (CIA) in vivo via intraarticular injection into the ankle joints bilaterally.

Results

In vitro experiments demonstrated that AdA20 suppressed NF‐κB activation, chemokine production, and matrix metalloproteinase secretion induced by tumor necrosis factor α in FLS. Mice with CIA that were treated with AdA20 had a lower cumulative disease incidence and severity of arthritis, based on hind paw thickness, radiologic and histopathologic findings, and inflammatory cytokine levels, than did control virus–injected mice. The protective effects of AdA20 were mediated by the inhibition of the NF‐κB signaling pathway. The severity of arthritis was also significantly decreased in the untreated front paws, indicating a beneficial systemic effect of local suppression of NF‐κB. Surprisingly, mice treated with AdA20 after the onset of CIA had significantly decreased arthritis severity from the onset of clinical signs to the end of the study.

Conclusion

These results suggest that using A20 to block the NF‐κB pathway in rheumatoid joints reduces both the inflammatory response and the tissue destruction. The development of an immunoregulatory strategy based on A20 may therefore have therapeutic potential in the treatment of RA.

     

Increased synovial tissue NF‐κB1 expression at sites adjacent to the cartilage–pannus junction in rheumatoid arthritis

Objective

To compare the expression of the Rel/NF‐κB subunits, NF‐κB1 (p50) and RelA (p65), in paired synovial tissue samples selected from sites adjacent to and remote from the cartilage–pannus junction (CPJ) in patients with inflammatory arthritis.

Methods

Synovial tissue was selected at arthroscopy from sites adjacent to the CPJ and from the suprapatellar pouch of patients who were referred to an early arthritis clinic. Tissue samples from patients with osteoarthritis (OA) undergoing knee arthroplasty were also studied. Rel/NF‐κB subunit activation and expression were measured by electrophoretic mobility shift assay and supershift analyses and by immunohistochemistry.

Results

Tissue samples were obtained from 10 patients with rheumatoid arthritis (RA), 7 with a seronegative arthropathy (SnA), and 6 with OA. Rel/NF‐κB was abundantly expressed in all samples. In both RA and SnA synovial tissue, the absolute number of NF‐κB1+ cells at the CPJ was significantly higher than at non‐CPJ sites (P = 0.006 and P = 0.02, respectively). The proportion of cells expressing NF‐κB1 was also significantly higher at the CPJ compared with non‐CPJ sites (P = 0.003 in RA, P = 0.009 in SnA). The numbers of RelA+ cells were consistently lower throughout. In RA synovial tissue, but not in SnA synovial tissue, both the absolute number and the proportion of RelA+ cells were significantly higher at the CPJ than at non‐CPJ sites (P = 0.003 and P = 0.01, respectively). In OA synovial tissue, the numbers of cells expressing NF‐κB1 and RelA were similar to those observed at the non‐CPJ sites in all inflammatory tissues studied.

Conclusion

In this study of early inflammatory arthritis, expression of NF‐κB1 in synovial tissue was highest at sites most likely to be associated with joint erosion. These observations are consistent with a critical role of NF‐κB1 in joint destruction, and support the rationale for specific therapeutic inhibition of NF‐κB in RA.

     

Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3

Objective

To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP‐3) in the pathology of early and late OA.

Methods

The role of synovial macrophages in MMP‐mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase‐induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP‐3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results

On day 14 following induction of OA, MMP‐mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean ± SD positively stained surface area 20 ± 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP‐induced neoepitopes was largely prevented (mean ± SD positively stained surface area 5 ± 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP‐mediated cartilage damage. We observed a strong decrease in MMP‐3 and MMP‐9 expression in synovial but not cartilage tissue in macrophage‐depleted joints. Among 2‐year‐old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP‐3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP‐3–knockout mice. In addition, MMP‐mediated VDIPEN expression was significantly decreased, indicating reduced MMP‐mediated cartilage breakdown.

Conclusion

The results of this study prove that MMP‐3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.

     

The role of TRAIL in mediating autophagy in myositis skeletal muscle: A potential nonimmune mechanism of muscle damage

Objective

Multinucleated cells are relatively resistant to classic apoptosis, and the factors initiating cell death and damage in myositis are not well defined. We hypothesized that nonimmune autophagic cell death may play a role in muscle fiber damage. Recent reports indicate that TRAIL may induce both NF‐κB activation and autophagic cell death in other systems. We undertook this study to investigate the role of TRAIL in cell death and pathogenesis in vitro and in vivo, using myositis muscle tissues from humans and mice.

Methods

Gene expression profiling was performed in myositis patient and control muscle specimens. Immunohistochemistry analysis was performed to confirm the gene array findings. We also analyzed TRAIL‐induced cell death (apoptosis and autophagy) and NF‐κB activation in vitro in cultured cells.

Results

TRAIL was expressed predominantly in myositis muscle fibers, but not in biopsy specimens from normal or other dystrophic‐diseased muscle. Autophagy markers were up‐regulated in humans with myositis and in mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NF‐κB activation and IκB degradation in cultured cells that are resistant to TRAIL‐induced apoptosis but that undergo autophagic cell death.

Conclusion

Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NF‐κB and autophagic cell death in myositis. Thus, this nonimmune pathway may be an attractive target for therapeutic intervention in myositis.

     

The peroxisome proliferator–activated receptor γ agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: In vivo protective effects mediated through the inhibition of key signaling and catabolic pathways

Objective

Emerging evidence indicates that peroxisome proliferator–activated receptor γ (PPARγ) may have protective effects in osteoarthritis (OA). The aim of this study was to evaluate the in vivo effect of a PPARγ agonist, pioglitazone, on the development of lesions in a canine model of OA, and to explore the influence of pioglitazone on the major signaling and metabolic pathways involved in OA pathophysiologic changes.

Methods

OA was surgically induced in dogs by sectioning of the anterior cruciate ligament. The dogs were then randomly divided into 3 treatment groups in which they were administered either placebo, 15 mg/day pioglitazone, or 30 mg/day pioglitazone orally for 8 weeks. Following treatment, the severity of cartilage lesions was scored. Cartilage specimens were processed for histologic and immunohistochemical evaluations; specific antibodies were used to study the levels of matrix metalloproteinase 1 (MMP‐1), ADAMTS‐5, and inducible nitric oxide synthase (iNOS), as well as phosphorylated MAPKs ERK‐1/2, p38, JNK, and NF‐κB p65.

Results

Pioglitazone reduced the development of cartilage lesions in a dose‐dependent manner, with the highest dosage producing a statistically significant change (P < 0.05). This decrease in lesions correlated with lower cartilage histologic scores. In addition, pioglitazone significantly reduced the synthesis of the key OA mediators MMP‐1, ADAMTS‐5, and iNOS and, at the same time, inhibited the activation of the signaling pathways for MAPKs ERK‐1/2, p38, and NF‐κB.

Conclusion

These results indicate the efficacy of pioglitazone in reducing cartilage lesions in vivo. The results also provide new and interesting insights into a therapeutic intervention for OA in which PPARγ activation can inhibit major signaling pathways of inflammation and reduce the synthesis of cartilage catabolic factors responsible for articular cartilage degradation.