Early intervention with Interleukin-1 Receptor Antagonist Protein modulates catabolic microRNA and mRNA expression in cartilage after impact injury A.A.

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Early intervention with Interleukin-1 Receptor Antagonist Protein modulates catabolic microRNA and mRNA expression in cartilage after impact injury A.A. Genemaras, T. Reiner, C.-Y. Huang, L. Kaplan Osteoarthritis and Cartilage Volume 23, Issue 11, Pages 2036-2044 (November 2015) DOI: 10.1016/j.joca.2015.05.010 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Calibration curve showing a linear relationship between grayscale image intensity and applied pressure. Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Example analysis of Fujifilm: typical medial condyle stain (A–C) and typical lateral condyle stain (D–F). A&D. Adjusted grayscale stain of digital raw stain (600 ppi) with averaged intensities calculated over areas 4 × 4 pixel areas and applied pressure distribution map. B&E. Colormap of pressures. C&F. Region identified in white with >20 MPa applied pressure. Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 (A,B) Relative expressions of inflammatory cytokines and catabolic enzymes (A) and expressions of miR-125b, miR-140, and miR-27b (B) at 3 and 8 h after impact injury. *indicates significance P < 0.05. Refer to text for exact P-value. (C–H) Immunohistochemical assessments of control and injured cartilage for expressions of IL-1β (D,G) and TNF-α (E,H). Negative control sections for control (B) and injured (F) cartilage showed negligible staining. Representative samples are shown. Original magnification × 200. Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Representative cell viability staining of cartilage at 8 h post-injury and IRAP treatment (10×) A. Control; B. Injury, No Treatment; C. Injury, IRAP treatment. Live cells (green), dead cells (red). Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Relative expressions of inflammatory cytokines, catabolic enzymes, and aggrecan (A) and expressions of miR-125b, miR-140, and miR-27b (B) in control, injured, and treated cartilage at 8 h after impact injury. *indicates significance P < 0.05. Refer to text for exact P-value. Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Relative expressions of inflammatory cytokines and catabolic enzymes (A) and expressions of miR-125b, miR-140, and miR-27b (B) in control, injured, and treated meniscus at 8 h after impact injury. *indicates significance P < 0.05. Refer to text for exact P-value. Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 A proposed mechanism of IRAP treatment. Impact injury causes up-regulation of IL-1β expression and secretion, which binds to the IL-1 receptor, incites the IL-1 catabolic pathway, and results in down-regulated miR-140 and miR-125b expressions and up-regulated aggrecanase expressions. IRAP prevents IL-1β from binding to the IL-1 receptor on the cell membrane and inhibits the paracrine and autocrine effects of IL-1β secreted. Therefore, expressions of miR-140 and miR-125b remain elevated due to mechanical loading and expressions of aggrecanases and inflammatory cytokines are down-regulated. Up-regulation or down-regulation of a specific gene is represented by an upward or downward arrow, respectively. All listed targets have been validated. The miRNA targets listed include aggrecanase-1 (ADAMTS-4), aggrecanase-2 (ADAMTS-5), and collagenase-3 (MMP-13). Osteoarthritis and Cartilage 2015 23, 2036-2044DOI: (10.1016/j.joca.2015.05.010) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions