The goal of this study was to determine the efficacy of local IL-1Ra gene therapy by intra-articular plasmid injections on structural changes in the meniscectomy rabbit model of osteoarthritis. A partial meniscectomy of the right knee was performed on the rabbits through a medial parapatellar incision. The rabbits were then divided into four experimental groups. Group 1 received no treatment. Group 2 received three consecutive intra-articular injections at 24-hour intervals of 0.9% saline containing a lipid, gammaAP-DLRIE/DOPE, and a DNA plasmid, VR1012. Group 3 received three consecutive injections of saline containing 1000 microg of canine IL-1Ra plasmid and lipid. The injections were given starting 4 weeks post-surgery. Rabbits from Group 1 were killed 4 weeks post-surgery, and all other rabbits 8 weeks post-surgery. The severity of macroscopic and microscopic changes on cartilage on the medial and femoral condyles and tibial plateaus and synovium were graded separately. Specimens were also processed for immunohistochemical staining using a rabbit polyclonal antibody against canine IL-1Ra. The level of canine IL-1Ra in synovial fluid was determined using enzyme-linked immunosorbent assay. The presence of the DNA plasmid in the synovium was tested by polymerase chain reaction. A significant reduction in the width of osteophytes and size of macroscopic lesions (P < 0.04) was observed, and was dependent on the amount of IL-1Ra plasmid injected. A significant reduction was also noted in the severity of histologic cartilage lesions (P < 0.01) in the group that received the highest dosage (1000 microg) of IL-1Ra plasmid. IL-1Ra was detected in synovial fluid by enzyme-linked immunosorbent assay and by immunohistochemical staining in the synovium and cartilage of rabbits that received injections containing the IL-1Ra plasmid. Polymerase chain reaction analysis of synovial DNA revealed the presence of the cloned cDNA dog IL-1Ra up to 4 weeks after the first intra-articular injection. This study demonstrates that direct in vivo transfer of the IL-1Ra gene into osteoarthritis knee cells using intra-articular injections of a plasmid vector and lipids can significantly reduce the progression of experimental osteoarthritis. This avenue may therefore represent a promising future treatment for osteoarthritis.

Objective. To examine, by immunohistochemistry, the localization and distribution of human collagenase-3 in normal, osteoarthritis (OA), and rheumatoid arthritis (RA) cartilage, and to investigate the effects of interleukin-lp (IL-1P) and transforming growth factor P (TGFP) on the synthesis and distribution of collagenase-3.Methods. Human cartilage specimens were obtained from tibia1 plateaus. In the first series of experiments, the OA specimens were excised from fibrillated and nonfibrillated areas of cartilage, and RA specimens were excised from lesional areas, including the cartilage-pannus junction when present. In the second series, full strips of cartilage were processed for culture in the presence or absence of IL-lP (100 units/ml) or TGFP (150 nglml). Each specimen was processed for immunohistochemical analysis using a collagenase-3 monoclonal antibody.Results. The number of cells that stained for collagenase-3 in normal cartilage was very low (-3%). In OA cartilage, the percentage increased dramatically, and no difference was found between fibrillated and nonfibrillated areas. A statistically significant increase Submitted for publication February 11, 1997; accepted in revised form April 29, 1997. in the percentage of cells staining for collagenase-3 was found in the deep layer compared with the superficial layer. This finding was noted in both the fibrillated areas (mean ? SEM 58.4 rt 1.6% and 40.1 f 3.9%, respectively; P < 0.007) and the nonfibrillated areas (55.4 rt 3.2% and 43.2 ? 2.7%; P < 0.01). Similarly, R4 cartilage showed a statistically significant (P < 0.001) increase in the level of chondrocytes staining positive for collagenase-3 in the deep layers (46.4 f 4.1%) compared with the superficial layers (26.2 rt 3.4%). In these RA specimens, the numbers of positively staining chondrocytes were similar both close to and a t a distance from the pannus junction. Both IL-lP and TGFP increased the number of chondrocytes producing collagenase-3. Interestingly, in normal specimens, TGFP had a predominant effect in the deep layers, while IL-1P had a greater effect on the superficial layers.Conclusion. This study demonstrates that, in situ, the increase in the level of chondrocytes synthesizing collagenase-3 in arthritic cartilage is predominant in the deep layers. The results further indicate that TGFP can up-regulate the level of this enzyme and, in normal cartilage in vitro, can cause a mimicking of the in situ distribution observed in arthritic cartilage.Articular cartilage is a very specialized connective tissue. It serves to provide the near-frictionless surfaces required for the smooth functioning of the joints, even under very highly loaded conditions. The cohesiveness of the extracellular matrix is maintained by the interaction between collagen fibrils and proteoglycans.Although osteoarthritis (OA) and rheumatoid arthritis (RA) are 2 distinct arthritic diseases, both

In the present study, we have investigated the effect of (i) ET-1 (endothelin-1) and its precursor, big ET-1, on MMP (matrix metalloproteinase)-2 and MMP-9 synthesis and activity in osteosarcoma tissue, and (ii) ET-1 receptor antagonists on cell invasion. Using Western blotting, zymography, RT-PCR (reverse transcription-PCR), immunohistochemistry, immunofluorescence and Northern blotting, we have shown that ET-1 and ET-1 receptors (ET(A) and ET(B)) were expressed in these cells. Additionally, we have demonstrated that ET-1 markedly induced the synthesis and activity of MMP-2, which was significantly increased when compared with MMP-9. Furthermore, inhibition of NF-kappaB (nuclear factor kappaB) activation blocked MMP-2 production and activity, indicating the involvement of NF-kappaB, a ubiquitous transcription factor playing a central role in the differentiation, proliferation and malignant transformation. Since ET-1 acts as an autocrine mediator through gelatinase induction and because inhibition of ET(A) receptor is beneficial for reducing both basal and ET-1-induced osteosarcoma cell invasion, targeting this receptor could be an attractive therapeutic alternative for the successful treatment of osteosarcoma.

Objective. The primary aim of this study was to investigate, using an experimental rabbit model of osteoarthritis (OA), the effect of a selective mitogenactivated protein kinase kinase 1/2 (MEK-1/2) inhibitor, PD 198306, on the development of structural changes. Additional aims were to assess the effects of the inhibitor on levels of phosphorylated extracellular signalregulated kinase 1/2 (phospho-ERK-1/2) and matrix metalloproteinase 1 (MMP-1; collagenase 1) in OA chondrocytes.Methods. After surgical sectioning of the anterior cruciate ligament of the right knee joint, rabbits with OA were separated into 3 experimental groups: oral treatment with placebo or with PD 198306 at a therapeutic concentration of 10 mg/kg/day or 30 mg/kg/day. Each treatment started immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopic and histologic studies were performed on the cartilage and synovial membrane. The levels of phospho-ERK-1/2 and MMP-1 in OA cartilage chondrocytes were evaluated by immunohistochemistry. Normal, untreated rabbits were used as controls.Results. OA rabbits treated with the highest dosage of MEK-1/2 inhibitor showed decreases in the surface area (size) of cartilage macroscopic lesions (P < 0.002) and in osteophyte width on the lateral condyles (P ‫؍‬ 0.05). Histologically, the severity of synovial inflammation (villous hyperplasia) was also reduced (P < 0.02). In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in the superficial layer stained positive for phospho-ERK-1/2 and MMP-1 compared with normal controls. Rabbits treated with the highest dosage of PD 198306 demonstrated a significant and dose-dependent reduction in the level of phospho-ERK-1/2 and a lower level of MMP-1.Conclusion. This study demonstrates that, in vivo, PD 198306, a selective inhibitor of MEK-1/2, can partially decrease the development of some of the structural changes in experimental OA. This effect was associated with a reduction in the level of phospho-ERK-1/2 in OA chondrocytes, which probably explains the action of the drug.