Fig. 1.

Impact of proteoglycan-4 (PRG4) and high-molecular weight hyaluronic acid (HA) treatments on TGF-β1-induced alpha smooth muscle actin (α-SMA) expression (ACTA2), production and stress fiber formation in fibroblast-like synoviocytes isolated from patients with OA (OA FLS) undergoing knee replacement surgery and CD44-dependent uptake of rhodamine-labeled recombinant human PRG4 (rhPRG4) into OA FLS and associated regulation of ACTA2 expression. OA FLS were treated with TGF-β1 (1 ng/mL) ± rhPRG4 (~ 240 kDa) or HA (~ 1200 kDa) (100 μg/mL for both treatments) for 24 h followed by RNA isolation and qPCR using GAPDH as an internal reference gene. OA FLS were stained with anti-α-SMA (green) and counterstained for α-tubulin (red) and DAPI (blue). Corrected total cell fluorescence (CTCF) of α-SMA was determined following a 48-h treatment, and normalized to controls. Rhodamine-labeled rhPRG4 was incubated with OA FLS ± anti-CD44 or isotype control (IC) antibodies for 30 min, and CTFC was quantified. OA FLS were treated with TGF-β1 ± rhPRG4 ± anti-CD44 or IC for 24 h and ACTA2 expression was determined. Data are presented as a scatterplot with means and standard deviations highlighted utilizing OA FLS from 3 to 4 different patients. *p < 0.001; ***p < 0.05. Scale = 50 μm. a PRG4 and HA reduced ACTA2 expression in OA FLS. b Representative images showing TGF-β1-induced stress fiber formation in OA FLS (white arrows) and PRG4 or HA treatments prevented their formation. c PRG4 and HA reduced α-SMA CTCF in OA FLS. d Representative images showing rhodamine-rhPRG4 intracellular localization (white arrows), following incubation with OA FLS. e Co-incubation of rhPRG4 and anti-CD44 reduced cellular uptake of rhPRG4. f Anti-CD44 antibody co-treatment reduced rhPRG4’s antifibrotic effect in OA FLS