Gene expression and protein analysis in ruptured human Achilles tendons. A comparison between ruptured and healthy area of the same tendon

Introduction: We studied the extracellular matrix (ECM) of 19 ruptured human Achilles tendons, comparing the tissue composition of specimens taken from area close to the rupture with specimens harvested from an apparently healthy area in the same tendon. Aim of this study was to analyze gene expression and ECM molecules as well as MMPs and TIMPs involved in ECM turnover, in order to asses the cellular activity and what might happen in Achilles tendon rupture. The hypothesis was that in the same tendon there are many differences in gene expression of ECM molecules and metalloproteinases activity between ruptured and macroscopically healthy areas. Materials and Methods: We compared the gene expression and the protein localization of the main ECM molecules (collagen type I and IX, decorin and versican) including enzymes involved in their metabolism as matrix metalloproteases (MMP2 and 9) and tissue inhibitory of metalloproteases (TIMP 1 and 2) using a real time RT-PCR, zymography and Fluorophore Assisted Carbohydrate Electrophoresis analysis. Results: We didn’t observe any collagen IX gene expression. The gene expression of collagen type I, proteoglycans GAGs, MMPs and TIMPs was more represented in the area close to the tendon rupture (p<0.05). The expression of MMPs was confirmed by zymography analysis, showing a marked increase of MMP9 activity in area close to the tendon rupture (p<0.05). The chemical composition of tendon changed showing that in the healthy area the GAGs content was higher than the ruptured area (p<0.05). Conclusions: The lack of gene expression of collagen IX testifies that there wasn’t any fibrocartilagineous metaplasia as described in tendinopathy. In the ruptured area, the tenocytes tried to restore the normal proteoglycan pattern increasing the protein synthesis but without the normal glycosaminoglycan production. The low amount of GAGs in the ruptured area indicates that the catabolic processes prevail over the synthetic activity. Our data support the hypothesis that, in human tendons, the tissue in the area of rupture undergoes marked rearrangement at molecular levels based on the MMP’s activity, and support the role of MMPs in the tendon pathology.