Damage mechanisms in tendon disease

Tendons are the thick fibrous cords of dense connective tissue that attach muscles to bone. As such, tendons play a critical role in nearly every musculoskeletal activity - from standing to running to writing. Research into tendon diseases lags far behind other areas of the musculoskeletal system.

Tendon injuries are a common cause of morbidity and significant health burden on society. One in three musculoskeletal GP consultations are the result of soft tissue tendon problem with an annual estimated cost to the NHS of £ 250 million. The current treatment available to doctors remains weak and as such a large proportion of patients remain under treated.

Our laboratory has highlighted a role for inflammation as a possible reason why some tendon injuries become chronic. We identified several damage-associated molecules including small inflammatory molecules (cytokines) that directly modify tendon tissue repair and worked to change tendon structure and cause it to be weaker. Subsequently we could block these molecules and improve the structure and function of the tendon. Importantly we have moved this discovery out of the laboratory and in a collaboration with industry partners aim to start treating real patents with tendon problems by 2019.

Evidence from other musculoskeletal diseases such as rheumatoid arthritis suggests that the cells present with your joints, fibroblasts, can become activated by inflammatory molecules and prevent patients from improving. In a similar manner, we and others have shown that the tendon cells, tenocytes, can produce inflammatory molecules when subjected to stress. In recent work, we have shown that tendon likely consists of several different types of tenocytes that are responsible for producing numerous inflammatory molecules that may either promote tendon healing or indeed further tendon damage.

This proposal aims to investigate whether different types of tendon cells can in fact modulate the inflammatory response that is seen in human tendon disease. Importantly since there are no approved drugs that directly target tendon cells, we anticipate that identifying different tendon cells that drive disease may reveal new drugs to reverse or at best stop further damage.

We will inform direct clinical care as follows:

1. Understanding how damage affects tendon cells at a cellular and molecular level.
2. Identifying if different tendon cells may be responsible for driving disease and how they can be modified to reverse tendon damage.

We will firstly explore the role of HMGB1 as a tenocyte subset DAMP effector pathway. Human tendon biopsies will be evaluated for HMGB1/TLR4 mRNA/protein expression followed by in vitro interrogation of the impact of exogenous HMGB1 on normal/diseased tendon via cytokine manipulations (rhHMGB1/anti HMGB1/TLR4 siRNA/HMGB1 chemical inhibitors) with the primary readout by RNAseq. Thereafter we will investigate the functional capacity of HMGB1 defined tenocyte subpopulations (FACS sorted HMGB1+/-) in vitro in the context of their ECM regulation (collagen synthesis/RT-PCR) and inflammatory potential (multiplex cytokine/chemokine assays) in tendon disease. Finally, to understand the net contribution of HMGB1 blockade on tendon injury we will utilise our previously published murine tendon injury model whereby neutralising antibodies to HMGB1/ HMGB1 inhibitors will be injected directly into the tendon immediately post injury in WT BALB/C mice. We will additionally carry out transfer adoptive transfer of HMGB1+/- tenocytes directly into WT injured tendon lesions with readouts as above and biomechanical assessment of tendon ex vivo.

Next,we intend to use two complimentary strategies to investigate tenocyte subpopulations obtained after tissue disaggregation.1)fluorescence sorting using a set of candidate protein markers followed by bulk transcriptomics (microarray/low input RNA seq) of gated populations. 2)unbiased single cell transcriptomics without gating-384 cell RNA sequencing to 6 independent tendon biopsy samples providing an in-depth transcriptional profile of tenocyte subpopulations across the spectrum of tendon pathology. Finally, we will investigate the functional capacity of subpopulations in vitro in the context of their ECM regulation/ inflammatory potential. Tenocyte subpopulations will be FAC-sorted by virtue of surface markers derived and undergo mechanical/inflammatory stimuli with primary readouts on measures of tenocyte function as detailed above.

This proposal is a basic science discovery project that aims to dissect fundamental processes in soft tissue immunobiology. In the short-term, therefore, the impact of the study will be most keenly felt in the academic community, and the principal beneficiaries will be immunology and musculoskeletal researchers in the UK and across the world. In addition, by communicating the research through our continuing teaching and outreach activities, we will educate and inform, with impact, other beneficiaries in the University, schools, and the local community. There may be possibilities for some commercialisation over the tenure of the grant, and we have experience of working with the private sector.

We also have a track record of translating findings in experimental animals into studies on humans, and have close links with clinical colleagues in Glasgow from a variety of different specialties. In an innovative approach, our group have characterised one of the key pathways whereby both humans and horses develop tendon disease. This has led to a new therapy (TenoMiR/ EquiMIR) for treating tendinopathy that is currently being trialled in horses with tendon disease. In collaboration with the world leading equine facility at Texas A&M University an experimental trial in equine disease has shown that a single injection of this therapy was able to significantly improve the resolution of tendon injury. We have applied high-level molecular interrogation to an under investigated yet highly prevalent and burdensome disease process. A ground breaking discovery of a single microRNA-dependent regulatory pathway in early tissue healing highlights a microRNA replacement therapy as a promising therapeutic option for human and equine tendon representing a major advance in patient and animal care. To this end 'Causeway Therapeutics' is a biotech spin out emerging from this innovative work.

These examples highlight our ambitions to engage effectively with companies and clinicians to enhance the impact of our work, and, if funded, we will continue to actively seek similar outputs from the proposed study over the course of the funding period.

Interfering with inflammatory/matrix crosstalk therefore clearly has therapeutic potential. We anticipate that our study will be of broad interest to pharmaceutical companies, particularly those seeking to repurpose drugs for the treatment of soft tissue injuries. In particular, our close collaboration with Pfizer, Eli Lily and Abbvie regarding small molecule kinase inhibitors as polycytokine inhibitors in inflammatory arthritis offers unique opportunities to target these pathways in soft tissue tendon disease. The economic, physical and psychological impact of tendon disease is enormous, and patients can struggle to lead normal lives, placing a heavy burden on health services and families alike. Any new developments in the treatment of these diseases would be most welcome and have a significant impact on the health, well being and economic contribution of patients. Whilst potential impacts are some way off, given our previous translational success we anticipate that the dissection of damage associated mechanisms within this proposal will deliver early phase human clinical trials within 5 years.