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. 2018 Jun 20;77(9):1372–1380. doi: 10.1136/annrheumdis-2018-212964

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© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/

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Connective tissue growth factor (CTGF) is covalently bound to latent TGFβ and is sequestered in the pericellular matrix (PCM) on heparan sulfate. (A) Rested porcine articular cartilage was treated with 100 ng/mL CTGF for 1 hour or 4 hours and the medium immunoblotted (under reducing conditions) for TGFβ. (B) Rested porcine cartilage was re-cut in fresh medium and the injury CM, at times specified, immunoblotted for CTGF and TGFβ (under reducing conditions). (C) Confocal microscopic images showing LAP1 and type VI collagen (ColVI) colocalising in the PCM of normal human articular cartilage, propidium iodide (PI). Scale bar, 10 µm. (D) Cartilage injury CM (1 hour) was electrophoresed under both reducing (+DTT) or non-reducing (−DTT) conditions and immunoblotted for LAP1 and CTGF. (E) CTGF was immunoprecipitated from injury CM using goat anti-CTGF antibody and immunoblotted for LAP1 (first panel) or CTGF (second panel) (under reducing conditions). * indicating bands for LAP1 and CTGF. (F) Cartilage explants were treated with or without 10 mU/mL heparitinase or chondroitinase for 4 hours. Medium was run under either non-reducing (−DTT) (F) or reducing (+DTT) (G) conditions and immunoblotted for LAP1, CTGF and TGFβ. (H) Culture medium was collected from isolated monolayer porcine chondrocytes over 24 hours and immunoblotted for CTGF or LAP under reducing or non-reducing conditions. ** shows weak 75 kDa band of LAP1. (I) Injury CM was treated with or without hydrochloric acid to calculate active and latent TGFβ1 protein levels by ELISA. Lower limit of detection, 125 pg/mL (n=3). (J) Porcine chondrocytes were treated (45 min) with or without injury CM pre-incubated with 1 µg/mL anti-TGFβ neutralising antibody or isotype control for 1 hour. Lysates were immunoblotted for pSMAD2 and tubulin.