Journal: Arthritis Research & Therapy

Article Title: Proteoglycan-4 regulates fibroblast to myofibroblast transition and expression of fibrotic genes in the synovium

doi: 10.1186/s13075-020-02207-x

Figure Lengend Snippet: 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

Article Snippet: Murine fibroblasts (NIH3T3; ATCC; 20,000 cells/well) were cultured on collagen type-I-coated 22-mm glass coverslips for 24 h in DMEM supplemented with 10% bovine calf serum (BCS).

Techniques: High Molecular Weight, Expressing, Isolation, Labeling, Recombinant, Staining, Fluorescence, Incubation, Control

Journal: Arthritis Research & Therapy

Article Title: Proteoglycan-4 regulates fibroblast to myofibroblast transition and expression of fibrotic genes in the synovium

doi: 10.1186/s13075-020-02207-x

Figure Lengend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on TGF-β induced stress fiber formation, vinculin expression, and formation of focal adhesions (FAs) in murine fibroblasts (NIH3T3) and its relationship to cell migration. Stress fiber formation was probed using an anti-alpha smooth muscle actin (α-SMA) antibody (green) and a blinded investigator evaluated % stress fiber-positive fibroblasts. Vinculin (a marker of FAs) expression was probed using an anti-vinculin antibody (green) and cells were counterstained using rhodamine-labeled phalloidin (cytoskeleton label; red). NIH3T3 Fibroblast migration was performed using a scratch assay and the wound closure percentage was determined. * p < 0 . 001 ; ** p < 0 . 01 ; *** p < 0 . 05 ; n.s., non-significant. Scale in a and f = 20 μm; a Representative images showing TGF-β induced stress fiber formation and vinculin staining (white arrows) in NIH3T3 fibroblasts. b rhPRG4 reduced stress fiber formation in NIH3T3 fibroblasts. c rhPRG4 reduced mean NIH3T3 fibroblast spread area. d rhPRG4 reduced the number of FAs in NIH3T3 fibroblasts. e rhPRG4 reduced the mean size of FAs in NIH3T3 fibroblasts. f Representative images showing NIH3T3 migration in response to TGF-β ± rhPRG4. g rhPRG4 reduced NIH3T3 migration

Article Snippet: Murine fibroblasts (NIH3T3; ATCC; 20,000 cells/well) were cultured on collagen type-I-coated 22-mm glass coverslips for 24 h in DMEM supplemented with 10% bovine calf serum (BCS).

Techniques: Recombinant, Expressing, Migration, Marker, Labeling, Wound Healing Assay, Staining

Journal: Arthritis Research & Therapy

Article Title: Proteoglycan-4 regulates fibroblast to myofibroblast transition and expression of fibrotic genes in the synovium

doi: 10.1186/s13075-020-02207-x

Figure Lengend Snippet: Stress fibers and focal adhesions (FAs) in murine synovial fibroblasts isolated from Prg4 −/− and Prg4 +/+ mice and their relationship to cell migration. Stress fiber formation was probed using an anti-alpha smooth muscle actin (α-SMA) antibody (green). Vinculin (a marker of FAs) expression was probed using an anti-vinculin antibody (green) and cells were counterstained using rhodamine-labeled phalloidin (cytoskeleton label; red). The impact of rhPRG4 treatment (200 μg/mL) on basal Prg4 −/− synovial fibroblast migration was assessed using the scratch assay and the wound closure percentage was determined. To highlight the CD44-dependency of the enhanced basal migration of Prg4 −/− synovial fibroblasts, cells were also treated with either an anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). * p < 0 . 001 ; ** p < 0 . 01 ; n.s., non-significant. Scale in a = 50 μm (α-SMA) and 100 μm (vinculin); scale in c = 50 μm. a Representative images showing increased stress fibers and FAs in Prg4 −/− synoviocytes. b Prg4 −/− synoviocytes demonstrated a higher mean FA size than Prg4 +/+ synoviocytes. c Representative images showing enhanced basal migration of Prg4 −/− synoviocytes compared to Prg4 +/+ synoviocytes. d rhPRG4 and anti-CD44 treatments were equally effective in reducing basal migration of Prg4 −/− synoviocytes

Article Snippet: Murine fibroblasts (NIH3T3; ATCC; 20,000 cells/well) were cultured on collagen type-I-coated 22-mm glass coverslips for 24 h in DMEM supplemented with 10% bovine calf serum (BCS).

Techniques: Isolation, Migration, Marker, Expressing, Labeling, Wound Healing Assay, Control

Journal: Arthritis Research & Therapy

Article Title: Proteoglycan-4 regulates fibroblast to myofibroblast transition and expression of fibrotic genes in the synovium

doi: 10.1186/s13075-020-02207-x

Figure Lengend Snippet: Impact of co-culturing murine fibroblasts (F) and murine macrophages (M) on active and total supernatant TGF-β levels, fibroblast migration, and efficacy of rhPRG4 in regulating fibroblast migration in the co-culture model. Murine macrophages (J774A) were stimulated with lipopolysaccharide (LPS; 5 μg/mL) for 24 h prior to seeding in the top chamber of a co-culture system. Murine fibroblasts (NIH3T3) were cultured in the lower chamber of the same system. Active and total TGF-β media levels were determined using an ELISA. A scratch was performed in the fibroblast monolayer and fibroblast migration in the lower chamber was determined at 48 h ± rhPRG4 (200 μg/mL). * p < 0 . 001 ; ** p < 0 . 01 ; n.s., non-significant. Scale = 40 μm. a Active and total TGF-β concentrations were higher in the fibroblast and macrophage co-culture compared to fibroblasts alone. b Representative images of fibroblast migration across different experimental groups. Fibroblast migration was highest in co-cultured fibroblasts and macrophages and rhPRG4 treatment reduced fibroblast migration. c rhPRG4 reduced fibroblast migration in a fibroblast and macrophage co-culture model. d rhPRG4 did not alter active or total TGF-β media levels in the fibroblast and macrophage co-culture model

Article Snippet: Murine fibroblasts (NIH3T3; ATCC; 20,000 cells/well) were cultured on collagen type-I-coated 22-mm glass coverslips for 24 h in DMEM supplemented with 10% bovine calf serum (BCS).

Techniques: Migration, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay