Trichostatin A regulates fibro/adipogenic progenitor adipogenesis epigenetically and reduces rotator cuff muscle fatty infiltration.

Rotator cuff (RC) muscle fatty infiltration (FI) is an important factor that determines the clinical outcome of patients with RC repair. There is no effective treatment for RC muscle FI at this time. The goal of this study is to define the role Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor in regulating muscle fibro/adipogenic progenitors (FAPs) adipogenesis and treating muscle fatty degeneration after massive RC tears in a mouse model. We hypothesize that TSA reduces muscle FI after massive RC tears. HDAC activity was measured in FAPs in RC muscle after tendon/nerve transection or sham surgery. FAPs were treated with TSA for 2 weeks and FAP adipogenesis was evaluated with perilipin and Oil Red O staining, as well as reverse transcript-polymerase chain reaction for adipogenesis-related genes. About 0.5 mg/kg TSA or dimethyl sulfoxide was administered to C57B/L6 mice with massive rotator cuff tears through daily intraperitoneal injection for 6 weeks. Supraspinatus muscles were harvested for biochemical and histology analysis. We found that FAPs showed significantly higher HDAC activity after RC tendon/nerve transection. TSA treatment significantly reduced HDAC activity and inhibited adipogenesis of FAPs. TSA also abolished the role of bone morphogenetic protein-7 in inducing FAP adipogenesis and promoted FAP brown/beige adipose tissue (BAT) differentiation. TSA injection significantly increased histone H3 acetylation and reduced FI of rotator cuff muscles after massive tendon tears. Results from this study showed that TSA can regulate FAP adipogenesis and promote FAP BAT differentiation epigenetically. HDAC inhibition may be a new treatment strategy to reduce muscle FI after RC tears and repair.

Leucine attenuates muscle atrophy and autophagosome formation by activating PI3K/AKT/mTOR signaling pathway in rotator cuff tears.

Rotator cuff tears (RCTs), the most common tendon injury, are always accompanied by muscle atrophy, which is characterized by excessive protein degradation. Autophagy-lysosome systems are the crucial proteolytic pathways and are activated in atrophying muscle. Thus, inhibition of the autophagy-lysosome pathway might be an alternative way to minimize skeletal muscle atrophy. In this present study, combined with a tendon transection-induced rat model of massive rotator cuff tears, HE staining and transmission electron microscopy methods, we found leucine supplementation effectively prevented muscle atrophy, muscle injury and autophagosome formation. Utilizing immunoblotting, we discovered that leucine supplementation is able to inhibit the rise in autophagy-related protein expression (including LC3, Atrogin-1, MuRF1, Bnip3 and FoxO3) driven by tendon transection. The PI3K/AKT/mTOR pathway that was essential in autophagosome formation and autophagy was blocked in atrophying muscle and reactivated in the presence of leucine. Once administrated with LY294002 (PI3K inhibitor) and Rapamycin (mTOR inhibitor), leucine mediated by the anti-atrophic effects was nearly blunted. These results suggest that leucine potentially attenuates tendon transection-induced muscle atrophy through autophagy inhibition via activating the PI3K/AKT/mTOR pathway.

Full-thickness rotator cuff tear in rat results in distinct temporal expression of multiple proteases in tendon, muscle, and cartilage.

The etiology of joint tissue degeneration following rotator cuff tear remains unclear. Thus, the purpose of this study was to understand the timeline of protease activity in the soft tissues of the shoulder (tendon, muscle, and cartilage) that may lead to down-stream degeneration following rotator cuff tear. A well-established rat model involving suprascapular nerve denervation and supraspinatus/infraspinatus tendon transection was employed. Histological staining and/or micro-computed tomography (µCT) were used to observe structural damage in the supraspinatus tendon and muscle, humeral head cartilage, and subchondral bone. Multiplex gelatin zymography was utilized to assess protease activity in the supraspinatus tendon and muscle, and humeral head cartilage. Zymography analysis demonstrated that cathepsins were upregulated in the first week in all tissues, while MMP-2 maintained prolonged activity in supraspinatus tendon between 1 and 3 weeks and increased only at 3 weeks in supraspinatus muscle. In supraspinatus tendon, increased cathepsin L and MMP-2 activity in the first week was concurrent with matrix disorganization and infiltration of inflammatory cells. In contrast, significant upregulation of cathepsin L and K activity in supraspinatus muscle and humeral head cartilage did not correspond to any visible tissue damage at 1 week. However, focal defects developed in half of all animals' humeral head cartilage by 12 weeks (volume: 0.12 ± 0.09 mm3 ). This work provides a more comprehensive understanding of biochemical changes to joint tissue over time following rotator cuff tear. Overall, this provides insight into potential therapeutic targets and will better inform ideal intervention times and treatments for each tissue. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:490-502, 2019.

Matrix Metalloproteases 1 and 3 Promoter Gene Polymorphism Is Associated With Rotator Cuff Tear.

BACKGROUND: Studies suggest that the collagen degeneration and disordered arrangement of collagen fibers in rotator cuff tears are associated with an increase in activity of matrix metalloproteases 1 and 3 (MMP-1 and MMP-3), and that MMP activity may be in part genetically mediated. The degree to which this might be clinically relevant in patients with rotator cuff tears has not been well characterized. QUESTIONS/PURPOSES: (1) Is genetic polymorphism of MMP-1 and MMP-3 associated with rotator cuff tears? (2) Are there haplotypes of MMP-1 and MMP-3 correlated with rotator cuff tears? (3) Compared with control subjects, do patients with rotator cuff tears have a higher proportion of relatives with the same disease? METHODS: We evaluated 64 patients with full-thickness rotator cuff tears and 64 asymptomatic control subjects. Patients younger 65 years, with nontraumatic tears, were included. The tear or integrity of the rotator cuff tear was evaluated by MRI or ultrasonography in all individuals. The patients and control subjects were paired by age. MMP-1 and MMP-3 genotypes were determined using the PCR-restriction fragment length polymorphism assays. RESULTS: Genetic polymorphisms in MMP-1 and MMP-3 are associated with rotator cuff tear, in which individuals with rotator cuff tears have associated genotypes 1G/2G (patients, 32 of 64 [50%], control subjects, 16 of 64 [25%]; odds ratio [OR], 4.8; 95% CI, 2.1-11.0; p < 0.001) and 2G/2G were at great risk (patients, 15 of 64 [23%], control subjects, seven of 64 [11%]; OR, 5.2; 95% CI,1.8-14.9; p < 0.001), and patients with rotator cuff tears were associated with a higher proportion of 2G allele distribution (62 of 128 [48%] versus 30 of 128 [23%]; p < 0.001). Patients with the 5A/5A genotype are at greater risk of rotator cuff tear (patients, 15 of 64 [23%]; control subjects, four of 64 [6%]; OR, 5.5; 95% CI, 1.4-20.9; p = 0.021), and there was higher 5A allele distribution in patients with rotator cuff tears (patients, 68 of 128 [53%]; control subjects, 52 of 128 [41%]; p = 0.045). Individuals with the haplotype 2G/5A were more likely to have rotator cuff tears develop (patients, 42 of 64 [66%]; control subjects, 17 of 64 [27%]; OR, 5.3; 95% CI, 2.5-11.3; p < 0.001). Patients with rotator cuff tears reported, in higher number, the existence of relatives who previously had treatment for rotator cuff tears (19 of 64 [30%] versus four of 64 [6%]; OR, 6.3; 95% CI, 2.0-19.9; p = 0.001). CONCLUSIONS: The genetic polymorphism of MMP-1 and MMP-3 is associated with rotator cuff tear. Individuals with haplotype 2G/5A were more susceptible to rotator cuff tears in the population studied. CLINICAL RELEVANCE: Knowledge of the genetic markers related to rotator cuff tears can enable identification of susceptible individuals and increase understanding of the pathogenesis of tendon degeneration.

Supraspinatus tendon overuse results in degenerative changes to tendon insertion region and adjacent humeral cartilage in a rat model.

The etiology of rotator cuff tendon overuse injuries is still not well understood. Furthermore, how this overuse injury impacts other components of the glenohumeral joint, including nearby articular cartilage, is also unclear. Therefore, this study sought to better understand the time course of tendon protease activity in a rat model of supraspinatus overuse, as well as determine effects of 10 weeks of overuse on humeral head articular cartilage. For these studies, multiplex gelatin zymography was used to characterize protease activity profiles in tendon and cartilage, while histological scoring/mechanical testing and micro-computed tomography (µCT) imaging were used to quantify structural damage in the supraspinatus tendon insertion and humeral articular cartilage, respectively. Histological scoring of supraspinatus tendon insertions revealed tendinopathic cellular and collagen fiber changes after 10 weeks of overuse when compared to controls, while mechanical testing revealed no significant differences between tensile moduli (overuse: 24.5 ± 11.5 MPa; control: 16.3 ± 8.7 MPa). EPIC-µCT imaging on humeral articular cartilage demonstrated significant cartilage thinning (overuse: 119.6 ± 6.34 µm; control: 195.4 ± 13.4µm), decreased proteoglycan content (overuse: 2.1 ± 0.18 cm-1 ; control: 1.65 ± 0.14 cm-1 ), and increased subchondral bone thickness (overuse: 216.2 ± 10.9 µm; control: 192 ± 17.8µm) in the overuse animals. Zymography results showed no significant upregulation of cathepsins or matrix metalloproteinases in tendon or cartilage at 2 or 10 weeks of overuse compared to controls. These results have further elucidated timing of protease activity over 10 weeks and suggest that damage occurs to other tissues in addition to the supraspinatus tendon in this overuse injury model. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1910-1918, 2017.