Novel biomarkers in regulating human dense connective tissue repair

Painful and degenerative disorders of the musculoskeletal system pose a tremendous burden on the healthcare system. One main reason is the limited knowledge of the underlying key factors and pathways responsible for the suboptimal and often varying healing outcomes of dense connective tissues (CTs), such as tendons, ligaments and knee meniscus, which have vital supportive, protective and force transmitting roles in the human body. The overall aim of this thesis was to identify novel biomarkers of dense CT healing. The thesis was designed to explore for biomarkers during the inflammatory- and proliferative healing phases in tissue biopsies and micro-dialysate, respectively, after Achilles tendon rupture (ATR) as a model of dense CT healing.

Using quantitative RT-PCR and immunohistochemistry of ATR tissue biopsies from the inflammatory healing phase the presence of potential biomarkers (Col I, Col III, FGF, FN, MMP-9) was identified. The gene expression of fibroblast growth factor (FGF) was positively associated with improved 1-year patient-reported- and functional outcomes. Higher Col III mRNA expression was associated with more perceived tendon strength at 1 year.

Proteomic profiling using quantitative mass spectrometry of ATR biopsies from the inflammatory healing phase disclosed 769 proteins, including 51 differentially expressed proteins among patients with good- versus poor 1-year outcome. Among them a novel biomarker, elongation factor-2 (eEF2), was identified as being strongly prognostic of the 1-year clinical outcome. Experimental exploration revealed that eEF2 regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis in dense CT healing.

Utilizing the proteomic profile from the inflammatory healing phase together with weighted co-expression network analysis a biomarker, inter-alpha-trypsin inhibitor heavy chain (ITIH4), associated with improved 1-year healing outcomes was discovered. Experimental explorations identified ITIH4 to stimulate collagen I production mediated by PPARγ signaling pathways.

Further proteomic profiling of micro-dialysate from the proliferative healing phase discovered 1288 unique proteins, whereof 9 upregulated, and 23 downregulated proteins in patients with good- versus poor 1-year outcome. Upregulated proteins were related mainly to extracellular matrix organization, while downregulated pathways were associated with functions such as thrombosis formation. The most reliable predictive biomarker was the downregulated pro-inflammatory complement factor D (CFD).

Expanded characterization of CFD demonstrated higher expression during inflammatory- and lower expression during proliferative healing phases in the good outcome patients. Further experimental explorations demonstrated that CFD improved repair by enhancing cell migration and collagen type I (Col1a1) production during inflammation, while exerting the opposite effects during proliferative healing.

The results of this thesis have established biomarkers eEF2, FGF, ITIH4 during the inflammatory healing phase and CFD mainly during the proliferative healing phase, all prognostic of improved patient outcome in tendon repair, presumably reflective of dense CT healing in general. These findings may lead to improved individualized treatment decisions, as well as accelerate the development of improved therapies to promote good long-term clinical healing outcomes.

List of scientific papers

I. J Chen, J Svensson, CJ Sundberg, AS Ahmed, PW Ackermann. FGF gene expression in injured tendons as a prognostic biomarker of 1-year patient outcome after Achilles tendon repair. J Exp Orthop. 8, 20 (2021).
https://doi.org/10.1186/s40634-021-00335-0

II. J Chen, J Wang, DA Hart, AS Ahmed, PW Ackermann. Complement factor D as a predictor of Achilles tendon healing and long-term patient outcomes. FASEB J. 36, e22365 (2022).
https://doi.org/10.1096/fj.202200200RR

III. J Chen, J Wang, X Wu, N Simon, CI Svensson, J Yuan, DA Hart, AS Ahmed, PW Ackermann. eEF2 improves connective tissue repair by regulating cellular death, autophagy, apoptosis, proliferation and migration. [Submitted]

IV. J Chen, J Wang, David A. Hart, AS. Ahmed, PW Ackermann. Complement factor D mediates human dense connecitive tissue repair by mediating collagen type I synthesis. [Manuscript]

V. X Wu, J Chen, W Sun, PW Ackermann, AS Ahmed. Network analysis identifies ITIH4 as a prognostic biomarker and therapeutic target of connective tissue regeneration. [Submitted]