Fig 1: Fap promotes OA progression in a mouse DMM model. a–c Safranin O/Fast Green staining in control and Fap KO mice treated with FAPi or vehicle after DMM surgery. Weekly intra-articular administration of FAPi (40 µg/kg body weight) or vehicle (PBS) in 10-week-old mice was started 3 days after the surgery and continued for 8 weeks before paraffin sectioning and safranin O/fast green staining of the knee joints. Representative cartilage erosion (a, top: femur, bottom: tibia), subchondral bone thickening (b) and synovitis (c) images are shown (n = 6–7 mice per genotype in each treatment group). Yellow dotted lines indicate the subchondral bone plate. Arrows indicate the synovium. Scale bars: 100 µm. d–f Quantification of the OARSI score (d), subchondral bone thickness (e) and synovitis score (f). OARSI scores were calculated based on the erosion of the medial tibial plateau cartilage (a, bottom). Subchondral bone thickness was measured as the mean distance of five evenly distributed measuring points between the lower edge of the articular cartilage and the roof of the cancellous bone. Synovitis scores were calculated by summing the enlargement of the synovial lining cell layer, density of the resident cells and inflammatory infiltration. g, h Immunostaining of Col II in the knee joints (g) with quantifications (h) (n = 6 mice per genotype in each treatment group). DAPI staining indicates the nucleus. Scale bars: 100 µm. The statistical significance was assessed using two-way ANOVAs with Sidak’s multiple comparison tests. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)
Fig 2: Pharmacological inhibition of Fap ameliorates joint symptoms after the onset of OA. a–c Safranin O/Fast Green staining in wild-type mice treated with FAPi or vehicle after DMM surgery. Weekly intra-articular administration of FAPi (40 µg·kg-1 body weight) or vehicle (PBS) was performed 4 weeks after DMM surgery in 10-week-old mice and continued for 8 weeks before paraffin sectioning and safranin O/fast green staining of the knee joints. Representative cartilage erosion (a, top: femur, bottom: tibia), subchondral bone thickening (b) and synovitis (c) images are shown (n = 6 mice per group). Yellow dotted lines indicate the subchondral bone plate. Arrows indicate the synovium. Scale bars: 100 µm. d–f Quantification of the OARSI score (d), subchondral bone thickness (e) and synovitis score (f). g, h Immunostaining of Col II in the knee joints (g) with quantification (h) (n = 6 mice per group). DAPI staining indicates the nucleus. Scale bars: 100 µm. The statistical significance was assessed using one-way ANOVAs with Tukey’s multiple comparison tests. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)
Fig 3: Oln inhibits OA progression in a Fap-dependent manner. a–c Safranin O/Fast Green staining in control, Oln KO and Oln/Fap double KO mouse knee joints 8 weeks after DMM surgery. DMM surgery was performed in 10-week-old mice. Representative cartilage erosion (a, top: femur, bottom: tibia), subchondral bone thickening (b) and synovitis (c) images are shown (n = 7–9 mice per genotype). Yellow dotted lines indicate the subchondral bone plate. Arrows indicate the synovium. Scale bars: 100 µm. d–f Quantification of the OARSI score (d), subchondral bone thickness (e) and synovitis score (f). g, h Immunostaining of Col II in the knee joints (g) with quantification (h) (n = 6 mice per genotype). DAPI staining indicates the nucleus. Scale bars: 100 µm. The statistical significance was assessed using one-way ANOVAs with Tukey’s multiple comparison tests. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)
Fig 4: Fap degrades denatured or MMP13-cleaved Col II. a Fap degrades denatured Col II in a dose-dependent manner. Denatured Col II (boiled at 95 °C for 10 min) was incubated with different amounts of rFap at 37 °C for 24 h. Samples were separated by SDS?PAGE and quantified by colloidal blue staining (n = 3 independent experiments). b Fap degrades denatured Col II in a time-dependent manner. Denatured Col II was incubated with rFap at 37 °C for 0–24 h (n = 3 independent experiments). c FAPi inhibits Fap-mediated degradation of denatured Col II. Different doses of FAPi were preincubated with rFap for 30 min before denatured Col II was added and further incubated at 37 °C for 24 h (n = 3 independent experiments). d Immunoprecipitation of Fap. HEK293T cells were transfected with GFP control, Oln-Flag, Fap-HA, or Oln-Flag + Fap-HA. Two days after transfection, Fap was immunoprecipitated from total cell lysates with 10 µL anti-HA affinity gel. Five percent total cell lysates were loaded as an input control (n = 3 independent experiments). e Oln inhibits the Fap-mediated degradation of denatured Col II. Col II was coincubated with immunoprecipitated samples (in 10 µL anti-HA affinity gel) at 37 °C for 24 h (n = 3 independent experiments). f Fap degrades MMP13-cleaved Col II. Native Col II was preincubated with rFap or rMMP13 at 37 °C for 12 h. EDTA was then added to the reaction mixture with or without rFap and incubated at 37 °C for another 12 h (n = 3 independent experiments). g Grayscale quantification of the 55, 40 and 30 kDa digestion bands in (f). h Experimental design. Primary chondrocytes were stimulated with vehicle control (PBS), 200 ng·mL-1 rFap, 200 ng·mL-1 rMMP13, or 200 ng·mL-1 rFap plus 200 ng·mL-1 rMMP13 for 24 h. i Western blot analysis of mouse Col2a1 protein levels in primary chondrocytes stimulated as in (h) (n = 3 independent experiments). The statistical significance was assessed using one-way ANOVAs with Tukey’s multiple comparison tests. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)
Fig 5: Expression analysis of Fap in OA synovium. a Immunostaining of human FAP in the synovium of control and OA patients. DAPI staining indicates the nucleus. Scale bars: 100 µm. b qPCR analysis of human FAP mRNA levels in the synovium of control and OA patients (n = 8 samples per group). c Western blot analysis of human FAP protein levels in the synovium of control and OA patients (n = 3 samples per group). d Immunostaining of mouse Fap in the knee joint of sham and DMM-treated mice. Sham or DMM surgery was performed in 8-week-old FapLacZ/+ mice, which were sacrificed 8 weeks later, and LacZ immunostaining was used to detect Fap expression at the posterior horn of the medial meniscus (F: femur; T: tibia; M: meniscus; S: synovium). DAPI staining indicates the nucleus. Scale bars: 100 µm. The statistical significance was assessed using two-tailed Student’s unpaired t tests. Data are presented as the mean ± SD (**P < 0.01)
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