3 research outputs found
Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.
OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA
The use of commercially available adhesive tapes to preserve cartilage and bone tissue integrity during cryosectioning
Increased Redox-Sensitive Green Fluorescent Protein Reduction Potential in the Endoplasmic Reticulum following Glutathione-Mediated Dimerization
As the endoplasmic reticulum (ER)
is the compartment where disulfide
bridges in secreted and cell surface proteins are formed, the disturbance
of its redox state has profound consequences, yet regulation of ER
redox potential remains poorly understood. To monitor the ER redox
state in live cells, several fluorescence-based sensors have been
developed. However, these sensors have yielded results that are inconsistent
with each other and with earlier non-fluorescence-based studies. One
particular green fluorescent protein (GFP)-based redox sensor, roGFP1-iL,
could detect oxidizing changes in the ER despite having a reduction
potential significantly lower than that previously reported for the
ER. We have confirmed these observations and determined the mechanisms
by which roGFP1-iL detects oxidizing changes. First, glutathione mediates
the formation of disulfide-bonded roGFP1-iL dimers with an intermediate
excitation fluorescence spectrum resembling a mixture of oxidized
and reduced monomers. Second, glutathione facilitates dimerization
of roGFP1-iL, which shifted the equilibrium from oxidized monomers
to dimers, thereby increasing the molecule’s reduction potential
compared with that of a dithiol redox buffer. We conclude that the
glutathione redox couple in the ER significantly increased the reduction
potential of roGFP1-iL <i>in vivo</i> by facilitating its
dimerization while preserving its ratiometric nature, which makes
it suitable for monitoring oxidizing and reducing changes in the ER
with a high degree of reliability in real time. The ability of roGFP1-iL
to detect both oxidizing and reducing changes in ER and its dynamic
response in glutathione redox buffer between approximately −190
and −130 mV <i>in vitro</i> suggests a range of ER
redox potentials consistent with those determined by earlier approaches
that did not involve fluorescent sensors