Investigating mechanisms driving fibrosis in pathological rotator cuff tendons

Shoulder pain is common, often caused by rotator cuff inflammation and usually heals by fibrotic repair. The fibrotic signature associated with the resolution of inflammation is well documented in a number of human tissue pathologies, but not tendon. TGFβ and its associated superfamily are implicated as key drivers of fibrosis, and modulate extracellular matrix homeostasis. The aim of this thesis was principally to investigate the TGFβ axis in healthy and pathological tendons, and to study changes related to the epithelial to mesenchymal transition. We hypothesised that TGFβ family mediators would be dysregulated in pathological compared to healthy tendons. We also hypothesised that there would be evidence of epithelial to mesenchymal transition with tendon disease. Healthy and diseased rotator cuff tendons were collected from patients presenting to an orthopaedic referral centre. Diseased tendinopathic (intact) and healthy rotator cuff tendons were collected via ultrasound-guided biopsy under general anaesthetic prior to surgery and torn tendons were collected during routine surgical debridement. Immunohistochemistry and qRT-PCR were used to investigate the protein and gene expression profiles of TGFβ superfamily members in these healthy and diseased rotator cuff tendons. Epithelial to mesenchymal transition was characterised with immunostaining for αSMA, E-Cadherin, and Vimentin. TLR4 and Collagen III were used to identify myofibroblast activation and collagen composition in tendon. TGFβ signalling was dysregulated in human supraspinatus tendon pathology. Specifically, TGFβ-1, TGFβ R1 and TGFβ R2 proteins were significantly reduced (p<0.01) in pathological compared to healthy supraspinatus tendons, suggesting these pathways become down regulated with established disease. At the mRNA level, TGFβ R1 was significantly decreased whereas TGFβ R2 was significantly increased (p<0.01). BMP-2, BMP-7 and CTGF mRNA were unchanged with pathology. Immunostaining showed Vimentin was significantly decreased in pathology (p<0.01), however αSMA, E-Cadherin and TLR4 remained unchanged. Pathological tendons showed dysregulation of the TGFβ axis compared to healthy tissues. Evidence for EMT in pathological tendons was inconclusive. Whilst Vimentin expression significantly decreased with pathology, immunostaining suggested co-localisation of Vimentin and αSMA along the edge of a massive tendon tear. We propose the down-regulation of TGFβ pathways could be a protective response to limit hypertrophic fibrosis. The disruption of the TGFβ axis with tendon pathology compared to healthy tissues demonstrates the importance of these pathways in tendon homeostasis. In the presence of suppressed TGFβ levels, alternative pathways such as TNFα signalling may drive fibrosis. In Dupuytren's disease TNFα is implicated as an important driver of myofibroblast activity, and could be the prevailing signal in chronic tendon pathology.