Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease that is characterized by synovial inflammation and bone erosion. We have investigated the mechanism(s) by which essential trace metals may initiate and propagate inflammatory phenotypes in synovial fibroblasts. We used HIG-82, rabbit fibroblast-like synovial cells (FLS), as a model system for potentially initiating RA through oxidative stress. We used potassium peroxychromate (PPC, Cr), ferrous chloride (FeCl Fe), and cuprous chloride (CuCl, Cu) trace metal agents as exogenous pro-oxidants. Intracellular ROS was quantified by fluorescence microscopy and confirmed by flow cytometry (FC). Protein expression levels were measured by western blot and FC, while ELISA was used to quantify the levels of cytokines. Trace metal agents in different valence states acted as exogenous pro-oxidants that generate reactive oxygen species (ROS), which signal through TLR4 stimulation. ROS/TLR4- coupled activation resulted in the release of HMGB1, TNF-α, IL-1β, and IL-10 in conjunction with upregulation of myeloid-related protein (MRP8/14) inflammatory markers that may contribute to the RA pathophysiology. Our results indicate that oxidant-induced TLR4 activation can release HMGB1 in combination with other inflammatory cytokines to mediate pro-inflammatory actions that contribute to RA pathogenesis. The pathway by which inflammatory and tissue erosive changes may occur in this model system possibly underlies the need for functioning anti-HMGB1-releasing agents and antioxidants that possess both dual trace metal chelating and oxidant scavenging properties in a directed combinatorial therapy for RA.
Reactive oxygen species (ROS) are implicated in playing a role in initiating and in propagating the pathogenesis of rheumatoid arthritis (RA). We investigated the mechanism(s) by which essential redox-active trace metals (RATM) may activate gene transcription in synovial fibroblasts. The rabbit fibroblast-like synovial cells which express Toll-like receptor 4 (TLR4), were used as a model system for potentially initiating RA through oxidative stress. Potassium peroxychromate (PPC, Cr5+), ferrous chloride (FeCl2, Fe2+), and cuprous chloride (CuCl, Cu+) at the indicated valency states were used as exogenous pro-oxidants. These trace metals can induce oxidative stress through TLR4 activation to release inflammatory cytokines and high mobility group box 1 protein. We measured the total expression levels of mitogen-activated protein kinase (MAPK) in the synovial cells and examined the effect of the redox-active trace metals on the time-course production of phosphorylated moieties of MAPK by fluorescence cell-sorting flow cytometry. TLR4 siRNA was used to examine the role of TLR4 in the activator protein -1 (AP-1) signalling activity, and western blots were used to measure the time-course phosphorylation levels of AP-1-activation-related proteins. While the redox-active trace metals increased intracellular ROS that can induce oxidative stress, they also induced MAPK kinases to upregulate the expression of AP-1 proteins in synovial cells. Our results show that redox-active trace metal/TLR4-coupled activation may contribute to the pathogenesis of RA. The signaling pathway by which inflammation and its destructive sequel may occur in RA through synovial cells underlies the need for developing therapeutic agents to serve in individualized RA therapy with a consideration for the underlying mechanism(s) of its pathogenesis.
Impaired myogenesis is linked to the muscle wasting and weakness characteristic of sarcopenia and muscular dystrophies. In the past, fusion index, a key biomarker of the myogenic differentiation process, has provided valuable data, but has required labor intensive techniques that often lack precision. Flow Cytometry (FC) provides an accurate and efficient way to characterize cell size, complexity and DNA content with forward scatter (FSC), side scatter (SSC), and propidium iodide staining (FL2), respectively. We propose that as myogenesis progresses, with the accompanying increase in fusion events, FC can be used to detect increasing mitochondrial biogenesis, DNA content and myosin heavy chain in C2C12 mouse skeletal myoblasts. For a more precise comparison of these parameters, we found it useful to synchronize cell cycle at G0/1 through serum deprivation for 24 hours. While still a developing methodology, our preliminary studies, demonstrate that FC could be a valuable method to quantify variations in cell size, character and DNA content in myoblasts and myotubes. This method promises to provide a more reliable characterization of myogenic differentiation to advance this field of research. Support: NIH‐National Institutes of Aging P01 AG039355 and Missouri Life Sciences Research Board (MB).
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