EARLY PATHOLOGICAL REMODELLING EVENTS, ACTORS AND REGULATORS IN OVERUSED SUPRASPINATUS TENDON

Introduction Tendon disorders due to overuse during professional activity, sport or ageing are a common cause of rheumatologic consultation and frequently affect the supraspinatus of the rotator cuff.1 Medical care comes often when tendinopathy has reached a chronic pain level or when rupture has occurred. Despite high social and financial cost of this pathology, the early pathological mechanism is not well known. Using a rat model of supraspinatus overuse,2 we aimed at deciphering the early events involved in extracellular matrix (ECM) remodelling in the tendon announcing a deleterious pathology in relationship with the inflammatory context. Methods Anabolic and catabolic biochemical analyses were performed on supraspinatus tendons from male Sprague-Dawley rats subjected to daily downhill treadmill running and control rats. Transcripts were measured by semi-quantitative RT-PCR and proteins determined by Western Blot. Total GAGs (glycosaminoglycans) were quantified using a DiMethyl Methylene blue (DMMB) based assay and specific amounts of Chondroitin Sulfate (CS), Dermatan Sulfate (DS) and Heparan Sulfate (HS) were determined by DMMB assay after combined pre-treatments of GAG extracts with nitrous acid and/or chondroitinase B. Immunohistochemistry on tendon sections allowed localising total GAGs by DMMB staining or specific PGs (proteoglycans) and GAGs with antibodies. Gelatinase activity of metalloproteinases (MMPs) was assayed by zymography and MMP inhibitors TIMP-1, TIMP-2 and TIMP-3 were detected with reverse gelatin zymography. MMP-2 and EMMPRIN/CD147 were detected in situ by immunolabelling. Inflammatory status was studied using a cytokine array and by immunodetection of pro-inflammatory an anti-inflammatory macrophages. Results After as soon as 2 weeks of tendon overuse, anabolic studies have shown collagen quantitative and qualitative alterations, increases in specific PGs and GAGs including aggrecan and CS, associated with an increase in the chondrogenic transcription factor sox9. These results proved that a precocious shift towards fibrocartilage occurs in the pure fibrous median zone of the tendon. Results also suggested that the cytokine HARP (Heparin Affine Regulatory Peptide) was implicated in this chondroid phenotypic change. In a catabolic study, MMP-2 and MMP-14/MT1-MMP appeared to be the sole enzyme candidates, among several MMPs tested, responsible for the observed matrix remodelling at 2 weeks. Whereas it appeared that remodelling events were not associated with inflammation in overused tendon, results suggested that the MMPs inducer EMMPRIN/CD147, which increased at 2 weeks of overuse and co-localized with MMP-2, is involved in MMP-14/MT1-MMP and MMP-2 up-regulation. Discussion This is the first report which shows specific GAGs (CS, DS, HS), together with PGs (decorin, versican, aggrecan), proteins alterations and localisation, complete with activation of specific MMPs and cytokines in early tendon overuse. These anabolic and catabolic alterations may temporarily interfere with the tendon9s tensile load-bearing capacity, which could lead to more deleterious damage. Anticipated detection of these early events, some of which may be reversible, could help to develop injury prevention strategies.