Tendon overuse, due to work- and sports-related activities, is the initiating factor for tendinopathy in the majority of patients, accounting for 30-50% of all sports medicine related injuries. The severity of subsequent impaired mechanical function, pain, and inflammation is influenced by age, activity level, genetic predisposition, co-morbities, and adverse drug effects. Due to the complexity of this musculoskeletal disease, successful pharmacological and physical therapies are still lacking. While the current model of pathogenesis includes published data on the nature of the initiating extrinsic factors (e.g. acute micro-injury and chronic overuse) and provides a molecular link between collagen fibril disorganization and loss of biomechanical function, it fails to provide any details on the nature of the cellular responses that produce a chronic non-functional tissue. The over-arching goal of this project was to advance the understanding of those cellular mechanisms involved in the initiation and progression of tendinopathy using epigenomic, transcriptomic, and proteomic methods.
Firstly, a murine model of Achilles tendinopathy was utilized to determine novel pathways associated with tendinopathy through epigenetic mechanisms (Aim 1). Methylome analyses in WT mice allowed for the discovery of differential methylation in the promoter regions of 5 genes (Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12) during the pathogenesis of tendinopathy. Notably their known functional roles are all relevant collagen dis-organization and development of chondroid metaplasia, typically associated with tendinopathy.
Histological evaluation of end stage diseased human tendons has suggested a potential involvement of hypoxia-mediated damage patterns to tendon cells and matrix, however, no molecular evidence has been reported to date. The murine Achilles tendinopathy model was used to examine the expression of a range of genes known to be affected by cellular responses to hypoxia (Aim 2). Overall expression levels of hypoxia signaling genes, specifically those involved in angiogenesis/coagulation and metabolism/transport were significantly altered with injury. Moreover, expression changes coincided with appearance of chondroid deposits in the pericellular and interfibrillar spaces of the tendon.
Lastly, given the changes in expression of genes involved in metabolism and those regulated by metabolism, a murine Achilles tendon explant system was developed to study the role of oxygen tension where intrinsic tendon cells could be studied within their native ECM (Aim 3). Injured explanted tendons demonstrated an inability to respond to changes in oxygen tension and exhibited altered metabolism (increased glucose uptake and NADH/NADPH production). All aims were also conducted with the Adamts5-/- mouse which exhibited a severe tendinopathic phenotype. In summary, using both in-vivo and ex-vivo murine systems, we have shown altered metabolic measures following tendon injury, which correlated with chondroid matrix deposition, a classic marker of tendinopathy. We postulate that altered tendon cellular metabolism following tendon injury may contribute to a chronic pathology and altered tendon healing.