Transglutaminase Modification of Tendon Collagen

CATEGORY: Ankle INTRODUCTION/PURPOSE: Achilles tendinopathy is one of the most common overuse conditions encountered in foot and ankle surgery. Various etiological risk factors for this disease have been explored: age, body-mass index, and patient specific biomechanics. However, the pathogenesis remains poorly understood. Healthy tendon is composed primarily of type III and I collagen, which undergo post-translationally modification by lysyl oxidase and transglutaminase (TG) enzymes. The expression and activity in the latter of these two is poorly characterized in Achilles tendon. This study aims to describe the function of a specific transglutaminase, TG2, within solubilized collagen and Achilles tenocyte cultures, and to demonstrate evidence of TG2 activity within pathologic human Achilles tendon tissue. METHODS: Acellular Collagen: Type I collagen gels were aliquoted into a silicone well plate. Gels were then seeded with either 2.4 ng of TG2 or a control. Two sets of gels were prepared; one undergoing a 10% stretch at 2Hz for 24 hours and the other a static control. TG2 substrates were labelled, and the relative quantity of higher molecular weight collagens produced by TG2 activity was compared. Tenocytes: Human tenocytes were grown in 3D cultures as well as traditional cultures in a conditioned fibroblast growth media. Proteins expressed by tenocytes in both cultures were extracted and examined for TG modification. Pathologic Tendon: After receiving Institutional Review Board approval, tissue was collected from patients undergoing debridement of Achilles tendinopathy. Collagen was extracted in 0.1M acetic acid and Western blot analysis was used to identify TG2 crosslink epitopes. RESULTS: In each of three experimental paradigms: acellular, cellular, and human tendon extracts, evidence for transglutaminase modification of collagen I was noted. Dynamic conditions increased TG2 modification of collagen in acellular cultures, as evidenced by higher molecular weight collagen forms. Western blot analysis of protein expression demonstrated that tenocytes cultured in 3-D produced higher levels of TG-modified proteins than those in 2-D. Similarly, western blot analysis demonstrated presence of TG2 modification epitopes in the harvested human Achilles tendon tissue. However, the enzyme itself did not appear to be strongly expressed and could not be detected from the samples. CONCLUSION: Transglutaminase 2 expression by tenocytes in 3D culture and evidence for transglutaminase-dependent modification of collagen I by TG2 under dynamic loading supports a key role for transglutaminase in tendon biology and in repair. Though evidence of TG2 modification was found in diseased human samples, the absence of detectable TG2 expression suggests that it may play a significant role in the healthy physiologic turnover of Achilles tendon collagen.