Faculty Bibliography

This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited ( approximately 35%, p = 0.02), and antibodies to F-spondin increased ( approximately 30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-beta treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-beta from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-beta dependent pathways.

Osteoarthritis (OA) is often a progressive and disabling disease, which occurs in the setting of a variety of risk factors--such as advancing age, obesity and trauma--that collude to incite a cascade of pathophysiological events within joint tissues. An important emerging theme in OA is a broadening of focus from a disease of cartilage to one of the 'whole joint.' The synovium, bone and cartilage are each involved in pathological processes that lead to progressive joint degeneration. Additional themes that have emerged over the past decade are novel mechanisms of cartilage degradation and repair, the relationship between biomechanics and biochemical pathways, the importance of inflammation and the role of genetics. In this article, we review the molecular, clinical and imaging evidence that synovitis is not an 'incidental finding of OA', but plays a significant role in disease pathogenesis, and could therefore represent a target for future treatments

T cell receptor (TCR)-dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is inhibited by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Protein kinase C-theta (PKC-theta) recruitment to the immunological synapse is required for full Teff activation. In contrast, PKC-theta was sequestered away from the Treg immunological synapse. Furthermore, PKC-theta blockade enhanced Treg function, demonstrating PKC-theta inhibits Treg-mediated suppression. Inhibition of PKC-theta protected Treg from inactivation by TNF-alpha, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-theta-mediated inhibition can function in the presence of inflammatory cytokines and thus have therapeutic potential in control of inflammatory diseases

BACKGROUND: A lack of biomarkers that identify patients at risk for severe osteoarthritis (OA) complicates development of disease-modifying OA drugs. OBJECTIVE: To determine whether inflammatory genetic markers could stratify patients with knee OA into high and low risk for destructive disease. METHODS: Genotype associations with knee OA severity were assessed in two Caucasian populations. Fifteen single nucleotide polymorphisms (SNPs) in six inflammatory genes were evaluated for association with radiographic severity and with synovial fluid mediators in a subset of the patients. RESULTS: Interleukin 1 receptor antagonist (IL1RN) SNPs (rs419598, rs315952 and rs9005) predicted Kellgren-Lawrence scores independently in each population. One IL1RN haplotype was associated with lower odds of radiographic severity (OR=0.15; 95% CI 0.065 to 0.349; p<0.0001), greater joint space width and lower synovial fluid cytokine levels. Carriage of the IL1RN haplotype influenced the age relationship with severity. CONCLUSION: IL1RN polymorphisms reproducibly contribute to disease severity in knee OA and may be useful biomarkers for patient selection in disease-modifying OA drug trials

MicroRNAs (miRNAs) represent a class of non-coding RNA molecules playing pivotal roles in cellular and developmental processes. miRNAs modulate the expression of multiple target genes at the post-transcriptional level and are predicted to affect up to one-third of all human protein-encoding genes. Recently, miRNA involvement in the adaptive and innate immune systems has been recognized. Rheumatoid arthritis serves an example of a chronic inflammatory disorder in which miRNAs modulate the inflammatory process in the joints, with the potential to serve as biomarkers for both the inflammatory process and the potential for therapeutic response. This review discusses the investigations that led to miRNA discovery, miRNA biogenesis and mode of action, and the diverse roles of miRNAs in modulating the immune and inflammatory responses. We conclude with a discussion of the implications of miRNA biology in rheumatoid arthritis and other autoimmune disorders

Background: The role of nitric oxide (NO) in osteoarthritis (OA) is controversial. NO, particularly when metabolized to peroxynitrite, is a recognized mediator of inflammation, which induces catabolic effects and promotes chondrocyte apoptosis. Conversely, NO delivered at low concentrations by NO donors exerts anti-inflammatory effects. Cyclooxygenase-Inhibiting Nitric Oxide Donators (CINODs) are anti-inflammatory compounds designed to inhibit both COX-1 and COX-2 while releasing nitric oxide, an important modulator of vascular tone. We studied the effect of naproxen and the CINOD NCX 429 in human chondrocytes and cartilage tissues from OA patients, to understand whether NO donation from CINODs may modulate the inflammatory/metabolic response. Methods: The experiments were performed with human cartilage and chondrocytes isolated from OA cartilage. Isolated chondrocytes stimulated with IL-1beta to induce inflammation, followed by measurement of inflammatory and matrix parameters, such as iNOS, COX-2, MMP1 and 13, PGE2, NO, matrix metalloproteinases, collagen degradation and NF-B nuclear translocation. Reference NSAIDs naproxen and celecoxib were used in comparison to NCX 429 (10 and 50 muM), and incubated 16 h before IL-1beta challenge. Cartilage explants culture supernatants (untreated and treated with modulators) were harvested at 24 and 72 hours post-treatment. RNA was extracted from chondrocytes and estimated by qPCR. NF-B binding was assessed using Marligen Bioscience kit and both cytoplasm and nuclear p65 subunit of NF-B was also assessed by Western blot. Results: The reference NSAIDs and the CINOD similarly upregulated MMP1 but inhibited MMP13, showing comparable net effect on collagen degradation as assayed by CTX-II ELISA. They similarly inhibited ADAMTS4, but only 50 muM NCX 429 downregulated COX-2 expression. In isolated OA chondrocytes, NCX 429 (10 and 50 muM) significantly and completely inhibited IL-1beta-induced PGE2 production, and both the constitutive and IL-1beta-stimulated induction of NF-B activity. Western blot analysis of p65 subunit isolated from nuclear fraction showed that the cells treated with CINOD (both in unstimulated and IL-1beta stimulated cells) had increased p65 accumulation although decreased NF-B binding and also inhibited NF-B promoter mediated luciferase reporter assay. Conclusions: Reference NSAIDs and the CINOD NCX 429 appear to similarly modulate human OA chondrocytes, both in unstimulated and stimulated conditions. NO has been considered detrimental for chondrocytes; however, donation of low concentrations of NO is recognized to be beneficial in a variety of conditions. Here, the CINOD NCX 429 does not differ from reference NSAIDs, indicating that CINODs do not adversely affect chondrocytes via NO donation. In addition, NCX 429 showed inhibition of the NF-B signalling, therefore presenting interesting anti-inflammatory features to be further explored