Abstract
BackgroundSynovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and is a major component of synovial fluid. PRG4 is a ligand of the CD44 receptor. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo.MethodsOA synoviocytes were treated with TGF-β ± PRG4 for 24 h and α-SMA content was determined using immunofluorescence. Rhodamine-labeled rhPRG4 was incubated with OA synoviocytes ± anti-CD44 or isotype control antibodies and cellular uptake of rhPRG4 was determined following a 30-min incubation and α-SMA expression following a 24-h incubation. HEK-TGF-β cells were treated with TGF-β ± rhPRG4 and Smad3 phosphorylation was determined using immunofluorescence and TGF-β/Smad pathway activation was determined colorimetrically. We probed for stress fibers and focal adhesions (FAs) in TGF-β-treated murine fibroblasts and fibroblast migration was quantified ± rhPRG4. Synovial expression of fibrotic markers: α-SMA, collagen type-I, and PLOD2 in Prg4 gene-trap (Prg4GT) and recombined Prg4GTR animals were studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-month-old Prg4GT/GT&Cd44−/− and Prg4GTR/GTR&Cd44−/− animals.ResultsPRG4 reduced α-SMA content in OA synoviocytes (p < 0.001). rhPRG4 was internalized by OA synoviocytes via CD44 and CD44 neutralization attenuated rhPRG4’s antifibrotic effect (p < 0.05). rhPRG4 reduced pSmad3 signal in HEK-TGF-β cells (p < 0.001) and TGF-β/Smad pathway activation (p < 0.001). rhPRG4 reduced the number of stress fiber-positive myofibroblasts, FAs mean size, and cell migration in TGF-β-treated NIH3T3 fibroblasts (p < 0.05). rhPRG4 inhibited fibroblast migration in a macrophage and fibroblast co-culture model without altering active or total TGF-β levels. Synovial tissues of 9-month-old Prg4GT/GT animals had higher α-SMA, collagen type-I, and PLOD2 (p < 0.001) content and Prg4 re-expression reduced these markers (p < 0.01). Prg4 re-expression also reduced α-SMA and PLOD2 staining in CD44-deficient mice.ConclusionPRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is partially mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo.
Highlights
Synovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I
We have reported that PRG4-Cluster determinant 44 (CD44) interaction inhibited interleukin-1 beta (IL-1β) induced OA Osteoarthritic fibroblast-like synoviocytes (FLS) proliferation and expression of matrix-degrading enzymes [40], via the inhibition of nuclear factor kappa b (NFκB) nuclear translocation mediated by blocking inhibitory kappa b (IκB) degradation [40]
PRG4 reduced Gene symbol for alpha smooth muscle actin (ACTA2) expression and stress fiber formation in osteoarthritic fibroblast-like synoviocytes (OA FLS) and CD44 was involved in the uptake of recombinant human proteoglycan-4 (rhPRG4) by OA FLS, whereas rhPRG4-CD44 interaction affected ACTA2 expression in response to TGF-β PRG4 and hyaluronic acid (HA) treatments demonstrated equivalent efficacies in reducing ACTA2 expression in OA FLS (Fig. 1a)
Summary
Synovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo. OA affects the whole joint with manifestations that include degeneration of articular cartilage and the meniscus, abnormal bone remodeling, and synovial inflammation [11,12,13]. Macroscopic evidence of synovial inflammation or synovitis is a common finding in up to 74% of patients with knee OA of different grades and 95% of patients with moderate to severe OA [14,15,16]. The extent of synovitis is a strong predictor of OA progression across multiple studies, and treating synovial inflammation is a potentially important target for therapeutic intervention, especially during the early stage of OA [14, 22,23,24,25,26,27]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
