"A well-balanced shift from pro- to anti-inflammatory processes during healing appears essential for successful regeneration and may prevent from chronic diseases or tissue damage. Resolution of inflammation is an active process, which is regulated by specialized pro-resolving mediators (SPMs) such as lipoxins, annexins, and resolvins. The contribution of these mediators to the severity of rotator cuff (RC) tendinopathy has previously been shown. Due to the close localization of RC tendons to the subacromial bursa, we hypothesize that bursae may store pro-resolving mediators to regulate tendon regeneration. We aimed to detect differences at increasing stages of RC tendinopathy in order to identify mediators that may regulate its deterioration.
We investigated bursae from healthy patients (n=13-16) versus patients with partial (n=13-17) or full-thickness RC tears (n=18-32). Bursa tissues were harvested for immunofluorescence staining, gene expression analysis and release of SPMs (Annexin A1, Lipoxin A4, Resolvin D1/D2) and/or their receptors (FPR2, ChemR23, GPR18). To understand regulatory mechanisms of SPM signaling in bursae, bursa-derived cells from patients with full-thickness RC tears (n=6) were subjected to physiological (2%) or pathological (8%) straining under uniaxial cyclic loading for 4 hours/day on 3 consecutive days. After stimulation, cells were analyzed by flow cytometry for SPM receptors (FPR2, ChemR23), fibroblast markers (CD90, CD105), adhesion molecules (CD54, CD106), human leucocyte antigens (HLA-DR, HLA-ABC) and proliferation marker Ki67. Furthermore, gene expression of SPM related genes (Annexin A1, FPR1, FPR2, ChemR23, GFP18), Col I, matrix degrading enzymes and inhibitors (MMP1, -2, TIMP1), and pro-inflammatory cytokines (IL6, IL1b) were analyzed.
Multiplex fluorescence staining revealed FPR2+ and ChemR23+ cells in perivascular, but also in fibrous or fatty bursa tissue. Single or double positive FPR2/ChemR23 cells were identified partially as CD45+ leukocytes but also as CD45- cells (e.g. endothelial cells, fibroblasts). Annexin A1 gene expression showed a trend towards an increase in bursae of partial RC tears compared to full-thickness RC tears (p=0.076), whereas SPM receptor gene expression was not significantly affected. In tissue culture, bursae of partial RC tears showed increased Annexin A1 and Resolvin D1 release compared to bursae of intact controls (p=0.029, p=0.007, respectively). Resolvin D1 release was also increased in bursae of full-thickness RC tears compared to intact controls (p=0.008). Pathological, but not physiological mechanical straining resulted in significantly increased MMP1 and ChemR23 gene expression compared to unstimulated controls (p=0.002, p=0.040). On surface marker level, no significant regulations were observed after mechanical stimulation of bursa-derived cells.
The study shows for the first time SPM signaling mediators in subacromial bursa tissue with differences depending on the severity of RC tears. The increase in Annexin A1 and Resolvin D1 particularly in early tendinopathy (partial RC tear) may indicate that these bursae are trying to balance the pro-inflammatory response at this stage. Pathological mechanical straining induced ECM remodeling, due to strongly increased MMP1 gene expression and might be an initiator of a pro-resolving response, as ChemR23 gene expression was up-regulated. In summary, the results provide first evidence that the subacromial bursa is involved in pro-resolving processes in RC tendinopathy."