Expression of the semicarbazide-sensitive amine oxidase in articular cartilage: its role in terminal differentiation of chondrocytes in rat and human

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Expression of the semicarbazide-sensitive amine oxidase in articular cartilage: its role in terminal differentiation of chondrocytes in rat and human A. Filip, A. Pinzano, A. Bianchi, B. Fève, S. Jalkanen, P. Gillet, D. Mainard, P. Lacolley, J. Magdalou, N. Mercier Osteoarthritis and Cartilage Volume 24, Issue 7, Pages 1223-1234 (July 2016) DOI: 10.1016/j.joca.2016.01.340 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Expression of SSAO in cartilage of normal rat. (A) Histological examination of cross section of Wistar rat knee joint stained with HES (morphology), safranin O (proteoglycan content), and immunostained with the anti-SSAO antibody. Only one representative picture is shown. Arrows indicate anti-SSAO immunostaining. The micrographs were taken at a magnification of 10× (subchondral bone and cartilage zone) and 40× (cartilage zone); the scale bars represent 200 μm. (B) SSAO immunostained of growth plate cartilage with a magnification of 10× and 40×. The scale bars represent 200 μm. (C) SSAO mRNA expression in articular cartilage and in positive controls (adipose tissue and aorta) from three rats. RPS29 mRNA expression was used for normalization. Results are expressed as fold change relative to aorta. (D) SSAO enzyme activity in articular cartilage, adipose tissue and aorta was expressed in nmol/h/mg. All results are presented as the mean value ± 95% CI of three different experiments, each performed in duplicate for real time qPCR and enzyme activity assay. All comparisons are made vs aorta values. Osteoarthritis and Cartilage 2016 24, 1223-1234DOI: (10.1016/j.joca.2016.01.340) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Expression of differentiation-dependent markers in primary cultures of rat chondrocytes. Rat chondrocytes were cultured from confluence (day 0) until day 21 in a differentiation medium. Terminal differentiation of chondrocytes was monitored at day 7, 14 and 21. (A) Representative photographs of chondrocytes in culture during the differentiation protocol. The micrographs were taken at magnification of 10×; the scale bar represents 200 μm. In these conditions, mRNA expression of chondrogenic markers: Sox 9 (B), collagen II (C), aggrecan (D) and mRNA expression of pre-hypertrophic collagen X (E) and hypertrophic markers MMP13 (F) and ALP (G) were quantified. RPS29 mRNA expression was used for standardization, and results are expressed as relative expression to day 0 expression according to delta delta Ct method. In graphs, dots represent the average of duplicate for a single cell culture. The lines show the average values ± 95% CI of three to four different cell cultures. Comparisons are made vs day 0 values. Osteoarthritis and Cartilage 2016 24, 1223-1234DOI: (10.1016/j.joca.2016.01.340) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Differentiation dependent expression and activity of SSAO in primary cultures of rat chondrocytes. (A) SSAO mRNA expression was evaluated by RT-qPCR. (B) SSAO protein expression was analyzed by western blotting in two different cell cultures. Immunoblot intensities for SSAO/Tubulin were quantified by densitometry. Intensities at days 0 were set at 1. A representative result is shown. (C) Enzyme activity was measured in parallel, and expressed in nmol/h/mg. In A and C graphs, dots represent means of duplicates and lines, the average value ± 95% CI of four to five different cell cultures. All comparisons are made vs day 0 values. Osteoarthritis and Cartilage 2016 24, 1223-1234DOI: (10.1016/j.joca.2016.01.340) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Chondrocyte viability and glucose transport. Chondrocytes were treated (gray triangles) or not (open circles) from confluence (day 0) with 1 μM LJP 1586, as a selective SSAO inhibitor. (A) Cell viability was determined by MTT reduction at days 0, 7, 14 and 21. Results are expressed as the difference between A560 and A620 nm the mean of four different cell cultures ± 95% CI. (B) Glucose transport was estimated by the entrance rate of 2-DG in chondrocyte, cultured 21 days after confluence as indicated in Methods. Lines indicate the mean ± 95% CI of five distinct cell cultures. Statistics were calculated with respect to control cells. When indicated, the brackets under P values indicate that LJP-treated values were compared vs control values of the corresponding time point. Osteoarthritis and Cartilage 2016 24, 1223-1234DOI: (10.1016/j.joca.2016.01.340) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of SSAO activity inhibition during the course of rat chondrocyte terminal differentiation. Chondrocytes were treated (black bars) or not (gray bars) from confluence (day 0) with 1 μM LJP 1586, as a selective SSAO inhibitor. (A) The SSAO enzyme activity expressed in nmol/h/mg was performed to confirm the efficacy of LJP 1586 in one experiment done in duplicate. The expression of SSAO (B), MMP13 hypertrophic marker (C), ALP (D) and OPN (E) mineralization markers and Collagen X pre-hypertrophic marker (F), were monitored by the determination of mRNA levels expressed as fold change relative to day 0, at days 7, 14 and 21, SSAO mRNA expression at days 7, 14 and 21, relative to day 0. Dots stent for one single cell culture performed in duplicate and lines represent the average value ± 95% CI of five to six different cell cultures. Statistics were calculated with respect to the day 0. The brackets under P values indicate that values from LJP-treated cells were compared vs control ones of the corresponding time point. Osteoarthritis and Cartilage 2016 24, 1223-1234DOI: (10.1016/j.joca.2016.01.340) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Expression of SSAO in human OA cartilage. The OA cartilage samples were obtained from knee joints of five patients. For each patient, a zone of preserved (Mankin score 2–4) or damaged cartilage (Mankin score 4 to 13) were taken for histology, SSAO activity and mRNA expression. (A) less affected or (B) damaged Human cartilage examination with HES (morphology), safranin O (proteoglycan content) staining and immunohistochemical analysis using anti-type X collagen and anti-SSAO antibodies. Representative micrographs of one patient were shown at a magnification of 10×, 20× and 40×. The scale bars represent 200 μm. SSAO enzyme activity in nmol/h/mg (C) and expression of collagen X (D), MMP13 (E), and OPN (F) mRNA in 2–4 Mankin score zone (open circles) and 4–13 Mankin score zone (gray triangles) cartilage were measured in five patients which means ± 95% CI are symbolized by lines. Results (D–F) are expressed as the relative expression of mRNA to the 2–4 score zone of each patient. Osteoarthritis and Cartilage 2016 24, 1223-1234DOI: (10.1016/j.joca.2016.01.340) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions