Poster 101: Evaluation of Novel Treatment Agents on Rotator Cuff Tendon and Muscle Pathology in a Murine Subacromial Impingement Model

OBJECTIVES: Rotator cuff disease is a prevalent disease process, and “molecular inflammation” through inflammatory mediators have been identified in pathologic process. Subacromial corticosteroid injections (CSIs) have been a mainstay of non-operative management for subacromial impingement; however, the specific inflammatory cell types and mediators which contribute to the process of tendinopathy have still not been well-elucidated. Previous work has demonstrated that TGF-b is involved in the development of fibrosis via immune cells and fibroblastic activity and is upregulated in a murine subacromial impingement model. Losartan, a commonly used antihypertensive drug, has been shown to reduce Tgfb1 expression and fibrosis development. Alpha-2 macroglobulin (A2M), a broad- spectrum MMP inhibitor, has been shown to increase collagen organization in a rat model of rotator cuff repair. Additionally, anakinra, an IL-1Ra, may mitigate soft tissue inflammation. The purpose of this study was to compare the effects of these three drugs, not previously utilized in the treatment of rotator cuff disease, with a CSI and with non-treated controls in the setting of subacromial impingement and rotator cuff tendinopathy. METHODS: All procedures were approved by our Institutional Animal Care and Use Committee. A total of 90 12-week-old male C57BL/6J mice were placed into 6 different groups (15 mice per group) (Fig 1A). Group 1 underwent a sham procedure with no treatment (sham controls). Group 2 underwent placement of a metal clip in bilateral shoulders to induce impingement on the rotator cuff but received no treatment. All mice receiving treatments (Groups 3-6) underwent bilateral clip impingement surgery on Day 0. At Day 21, treatments were initiated (Fig 1B). Subacromial injection of medications was done under direct visualization of the subacromial space and mice in the losartan group received the drug systemically in their drinking water (Fig 1A). All mice were sacrificed at 6 weeks following the initial impingement surgery. The supraspinatus tendons and muscles were harvested. Histology, biomechanical testing, flow cytometry, and gene expression analysis were performed as outcome measures. H&E, Alcian Blue, PSR, and Factor VIII IHC stains were performed for tendon and a modified Bonar score was calculated by two blinded scorers. Muscle samples were stained with H&E and Oil Red O. Biomechanical testing was performed using a customized MTS setup. Whole tendon samples were enzymatically digested and cells were stained for flow cytometry using a 20-color panel to identify both immune and stromal cells. For gene expression analysis, RNA isolation was performed for both tendon and muscle samples and was analyzed using the NanoString nCounter® Fibrosis Panel. The significance level was set at p=0.05 for all statistical analyses. RESULTS: There were no significant differences between treatment groups in tendon histology Bonar scores (Groups 3-6) for the supraspinatus tendons, and the scores were similar to historical controls of untreated subacromial impingement tendons. There were no differences in the biomechanical properties of the tendons between any of the impingement groups (2-6). Muscle histology demonstrated no differences in muscle fiber cross sectional area between groups, but there was a significantly higher area fraction of fat in the muscles of the CSI-treated group compared to the sham control, anakinra, and losartan treated groups (Fig 1C-E). NanoString analysis of muscle samples demonstrated higher mean pathway scores for de novo lipogenesis, fatty acid metabolism, and PPAR in the CSI-treated group, but this did not reach statistical significance. On flow cytometry, there were similar immune cell profiles between all groups (1-6). The only exception was there was a higher percentage of B cells in the anakinra-treated group compared to the impingement control group (3.38% vs. 0.88%, p<0.05) (Fig 2A-C). Treatment groups (particularly groups 4-6) exhibited individual stromal marker profiles more similar to the sham controls than to the impingement control groups with significantly higher percentages of CD51+, CD73+, CD90+, and CD105+ cells compared to the impingement control group (Fig 2D-G). Gene expression analysis of tendon samples demonstrated significantly decreased pathway scores for cytokine signaling, inflammasome, phagocytic cell function, oxidative stress, and proteotoxic stress in several of the treatment groups compared to the impingement control group (Fig 3A-E). Additionally, the CSI-treated group only demonstrated a significantly decreased pathway score for cytokine signaling but not for the other pathways compared to the impingement control group, suggesting that some of these novel treatments may have promise in mitigating the inflammatory and stress responses in the setting of subacromial impingement. CONCLUSIONS: Despite the prevalence of rotator cuff disease and our growing understanding of the inflammatory processes involved in tendinopathy, CSIs have remained a commonly utilized intervention in the non-operative management of subacromial impingement. We report here that three novel therapeutic agents (A2M, anakinra, and losartan) may have utility in promoting a favorable environment for stromal cells and for decreasing cytokine signaling, inflammatory responses, and stress pathways associated with subacromial impingement. While no gross structural tendon differences were found in this study, the cell phenotype and transcriptional profiling data suggest that further investigation into these agents and the underlying mechanisms of tendinopathy-related inflammation may allow for the development of alternatives to CSIs, which have known deleterious effects.