WISP1/CCN4 aggravates cartilage degeneration in experimental osteoarthritis  M.H. van den Bosch, A.B. Blom, V. Kram, A. Maeda, S. Sikka, Y. Gabet, T.M. Kilts, W.B. van den Berg, P.L. van Lent, P.M. van der Kraan, M.F. Young  Osteoarthritis and Cartilage  Volume 25, Issue 11, Pages 1900-1911 (November 2017) DOI: 10.1016/j.joca.2017.07.012 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 No decrease in spontaneous cartilage damage upon aging in the absence of WISP1. Wisp1−/− mice were generated and aged up to 18 months together with WT (Wisp1+/+) controls. Cartilage degeneration was assessed after 3, 6, 12, and 18 months. Whereas cartilage damage increased with age, no differences were observed between WT and Wisp1−/− mice at 3, 6, 12, and 18 months of age. Photomicrographs (original magnification ×100) of representative Safranin-O/Fast Green stained sections of 3 months old WT and Wisp1−/− mice. Univariate scatter plots are shown, with horizontal and vertical lines showing median ± interquartile range. Osteoarthritis and Cartilage 2017 25, 1900-1911DOI: (10.1016/j.joca.2017.07.012) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Reduced cartilage degeneration during experimental OA models in Wisp1−/− mice. Representative photomicrographs (original magnification ×100) are shown from Safranin-O/Fast Green-stained sections of knee joints, after induction of CIOA, DMM or ACLT in WT (Wisp1+/+) and Wisp1−/− mice. Arrowheads point to areas with cartilage degeneration. At 42 days after induction of CIOA, significantly decreased cartilage degeneration was observed at the medial side of the tibia, medial side of the femur and when the mean was calculated of these cartilage areas (A). Induction of the DMM experimental OA model (B) and the ACLT model (C) resulted in significantly decreased cartilage damage in Wisp1−/− mice at 56 days after induction. Univariate scatter plots are shown, with horizontal and vertical lines showing median ± interquartile range. Osteoarthritis and Cartilage 2017 25, 1900-1911DOI: (10.1016/j.joca.2017.07.012) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Increased cortical thickness and reduced trabecular spacing but no differences in osteophyte formation in Wisp1−/− mice. X-ray analysis after induction of the DMM model suggested a more uneven joint space width in WT mice (Wisp1+/+) compared to Wisp1−/− mice. Histology and X-rays suggested differences in the bone compartment between WT and Wisp1−/− mice (A). A more detailed investigation of the surface area using histology showed comparable sizes of osteophyte formation on the medial side of the femur and tibia in WT and Wisp1−/− mice (B). MicroCT analysis showed no differences in bone volume/total volume and bone mineral density of the entire subchondral bone (C) while the Wisp1−/− mice showed an increased cortical thickness (D) and reduced trabecular spacing (E), compared to WT mice. Representative three-dimensional reconstructions of the subchondral bone of the tibia are shown (F). Univariate scatter plots are shown, with horizontal and vertical lines showing median ± interquartile range. Representative X-rays and photomicrographs of Safranin-O/Fast Green-stained sections (A) and three-dimensional reconstructions (F) are shown. M = calcified and dislocated meniscus; O = osteophyte; S = subchondral bone; J = joint space. Osteoarthritis and Cartilage 2017 25, 1900-1911DOI: (10.1016/j.joca.2017.07.012) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Decreased expression of MMPs and ADAMTSs during experimental OA in mice lacking WISP1. CIOA was induced in WT (Wisp1+/+) and Wisp1−/− mice and 7 days after induction of the model, total RNA was isolated from synovial tissue biopsies. Reduced expression of Mmp3 and Mmp9, and Adamts4 and Adamts5 was observed in Wisp1−/− mice compared to WT controls, as determined by qRT-PCR. Additionally, the expression of Timp1 was decreased in Wisp1−/− mice. The expression of Timp3, which is known to be a potent inhibitor of ADAMTS4 and ADAMTS5 was not different between WT and Wisp1−/− mice (A). In contrast, no differences in gene expression between WT and Wisp1−/− mice were found in the articular cartilage (B). Univariate scatter plots are shown, with horizontal and vertical lines showing mean ± sem. Osteoarthritis and Cartilage 2017 25, 1900-1911DOI: (10.1016/j.joca.2017.07.012) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Reduced protease activity is observed in the cartilage of Wisp1−/− mice. Protease activity in the cartilage during the CIOA model was determined by immunohistochemical staining for the aggrecan neoepitope NITEGE. Representative micrographs (original magnification ×400) of NITEGE-stained sections and the isotype IgG control are shown (A), which suggest that the amount of protease activity is decreased in mice lacking Wisp1 expression compared to WT (Wisp1+/+) controls, in both damaged and relatively unaffected parts of the cartilage. Quantitative scoring of the positively stained area for three sections per joint confirmed that the protease activity was significantly decreased in Wisp1−/− under these circumstances (B). Univariate scatter plots are shown, with horizontal and vertical lines showing median ± interquartile range. Osteoarthritis and Cartilage 2017 25, 1900-1911DOI: (10.1016/j.joca.2017.07.012) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Increased protease expression in human end-stage OA synovium after stimulation with WISP1. mRNA expression levels of MMP3, MMP9, MMP13 and ADAMTS4 in end-stage human OA synovial tissue were significantly increased after stimulation with 500 ng/mL recombinant human WISP1 for 24 h compared to non-stimulated (NS) controls (A). Protein levels of MMP1 and MMP9 in the culture supernatant, as determined by Luminex multiplex analysis, were significantly increased after stimulation of end-stage OA synovium with WISP1 (B). Univariate scatter plots are shown, with horizontal and vertical lines showing mean ± sem. Osteoarthritis and Cartilage 2017 25, 1900-1911DOI: (10.1016/j.joca.2017.07.012) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions