Reduced response of human meniscal cells to Osteogenic Protein 1 during osteoarthritis and pro-inflammatory stimulation.

OBJECTIVE: Many cell types lose responsiveness to anabolic factors during inflammation and disease. Osteogenic Protein 1 (OP1/BMP7) was evaluated for the ability to enhance extracellular matrix synthesis in healthy and OA meniscus cells. Mechanisms of cell response to OP1 were explored. DESIGN: Meniscus and cartilage tissues from healthy tissue donors and osteoarthritis (OA) patients undergoing total knee arthroplasties were acquired. Primary cell cultures were stimulated with OP1 and/or inflammatory factors (IL1α, IL1ß, or fibronectin fragments (FnF)) and cellular responses were analyzed by RT-qPCR and immunoblots. Frozen section immunohistochemistry (IHC) was conducted to assess OP1 and receptor proteins in normal and OA meniscus. RESULTS: OP1 treatment of normal meniscus cells resulted in significant, dose-dependent increases in ACAN (aggrecan) and COL2A1, and decreased MMP13 gene transcription, while only ACAN was upregulated (P < 0.01) at the highest dose of OP1 in OA meniscus cells. OP1 induced significantly more ACAN gene transcription in normal meniscus than normal articular cartilage (P = 0.05), and no differences between normal and OA cartilage were detected. Receptor expression and kinetics of canonical signaling activation were similar between normal and OA specimens. Normal meniscus cells treated with inflammatory factors were refractory to OP1 stimulation. Smad1 phosphorylation at an inhibitory site was induced (P = 0.01 for both normal and OA meniscus) by inflammatory cytokine treatment. CONCLUSIONS: The meniscus demonstrates resistance to OP1 stimulation in OA and in the presence of inflammatory mediators. MAPK-mediated Smad1 linker phosphorylation is a possible mediator of the loss of anabolic extracellular matrix production in the inflammatory cytokine affected meniscus.

Osteoarthritic changes in vervet monkey knees correlate with meniscus degradation and increased matrix metalloproteinase and cytokine secretion.

OBJECTIVE: Meniscus injury increases osteoarthritis risk but its pathobiology in osteoarthritis is unclear. We hypothesized that older adult vervet monkeys would exhibit knee osteoarthritic changes and the degenerative menisci from these animals would secrete matrix metalloproteinases (MMPs) and pro-inflammatory cytokines that contribute to the development of osteoarthritis. DESIGN: In a cross sectional analysis of healthy young adult (9-12 years) and old (19-26 years) adult female vervet monkeys, knees were evaluated in vivo with computed tomography (CT) imaging, and joint tissues were morphologically graded at necropsy. Meniscus explants were subsequently cultured to evaluate meniscal MMP and cytokine secretion. RESULTS: CT images revealed significant bony osteoarthritic changes in 80% of older monkeys which included increases in osteophyte number and meniscal calcification. Meniscus and cartilage degradation scores were greater in the older monkeys and were positively correlated (r > 0.7). Menisci from older animals exhibiting osteoarthritic changes secreted significantly more MMP-1, MMP-3, and MMP-8 than healthy menisci from younger monkeys. Older menisci without significant osteoarthritic changes secreted more IL-7 than healthy young menisci while older osteoarthritic menisci secreted more IL-7 and granulocyte-macrophage colony-stimulating factor than healthy older menisci. CONCLUSIONS: Aged vervets develop naturally occurring knee osteoarthritis that includes involvement of the meniscus. Degenerative menisci secreted markedly increased amounts of matrix-degrading enzymes and inflammatory cytokines. These factors would be expected to act on the meniscus tissue and local joint tissues and may ultimately promote osteoarthritis development. These finding also suggest vervet monkeys are a useful animal model for studying the progression of osteoarthritis.

Pro-inflammatory stimulation of meniscus cells increases production of matrix metalloproteinases and additional catabolic factors involved in osteoarthritis pathogenesis.

OBJECTIVE: Meniscus injury increases the risk of osteoarthritis; however, the biologic mechanism remains unknown. We hypothesized that pro-inflammatory stimulation of meniscus would increase production of matrix-degrading enzymes, cytokines and chemokines which cause joint tissue destruction and could contribute to osteoarthritis development. DESIGN: Meniscus and cartilage tissue from healthy tissue donors and total knee arthroplasties (TKAs) was cultured. Primary cell cultures were stimulated with pro-inflammatory factors [IL-1ß, IL-6, or fibronectin fragments (FnF)] and cellular responses were analyzed by real-time PCR, protein arrays and immunoblots. To determine if NF-κB was required for MMP production, meniscus cultures were treated with inflammatory factors with and without the NF-κB inhibitor, hypoestoxide. RESULTS: Normal and osteoarthritic meniscus cells increased their MMP secretion in response to stimulation, but specific patterns emerged that were unique to each stimulus with the greatest number of MMPs expressed in response to FnF. Meniscus collagen and connective tissue growth factor (CTGF) gene expression was reduced. Expression of cytokines (IL-1α, IL-1ß, IL-6), chemokines (IL-8, CXCL1, CXCL2, CSF1) and components of the NF-κB and tumor necrosis factor (TNF) family were significantly increased. Cytokine and chemokine protein production was also increased by stimulation. When primary cell cultures were treated with hypoestoxide in conjunction with pro-inflammatory stimulation, p65 activation was reduced as were MMP-1 and MMP-3 production. CONCLUSIONS: Pro-inflammatory stimulation of meniscus cells increased matrix metalloproteinase production and catabolic gene expression. The meniscus could have an active biologic role in osteoarthritis development following joint injury through increased production of cytokines, chemokines, and matrix-degrading enzymes.

Brother of CDO (BOC) expression in equine articular cartilage.

Brother of CDO (BOC) is a cell surface receptor that derives its name from the structurally related protein, cell adhesion molecule-related/down-regulated by oncogenes (CDO, sometimes CDON). High levels of BOC mRNA and protein expression have been described in embryonic tissues with active cell proliferation and ongoing cellular differentiation(1,2). A microarray-based screen of RNA isolated from 11 different adult equine tissues unexpectedly identified BOC as having an expression pattern restricted to articular cartilage. The objective of this study was to further investigate BOC expression in adult articular cartilage relative to other tissues. Both RT-qPCR and mRNA sequencing confirmed the microarray data. Steady state BOC mRNA levels in articular cartilage were substantially higher than in the other adult tissues tested, neonatal tendon, placenta, and whole embryo. The expression of BOC displayed a pattern of tissue specificity comparable to well established cartilage matrix protein biomarkers. BOC mRNA levels in articular cartilage increased with age, but were rapidly down-regulated when chondrocytes were enzymatically isolated from the cartilage matrix and expanded in monolayer culture. Relative expression patterns of CDO were broadly similar, but displayed lower fold change differences. A functional role in articular cartilage that involves Hedgehog signaling is suggested by the known binding affinity of BOC for all three Hedgehog ligands. These data also extend BOC and CDO biology to a post-mitotic and highly differentiated cell type within a mature tissue.