H&E staining of biceps tendons—Group-1 (Patient numbers—PT3, PT6, PT8 and PT10) and Group-2 (Patient numbers–PT2, PT4, PT13 and PT9).

<p>The green arrows show tendon cells; black arrows point inflammation; red arrows indicate angiogenesis; blue arrows show ECM disorganization; violet arrows point fatty infiltration; and yellow arrows indicate normal ECM with dense collagen deposition. The inflammation and fatty infiltration were not evident in Group-2 while ECM disorganization was less prominent compared to Group-1. The figures are shown in 400x magnification.</p

Flow cytometry analysis of TREM-1 and TREM-2 expression on CD16⁺ and CD14⁺ cells.

<p>(A) Representative images for the gating of the cells in Group-1 and Group-2 patients. (B) Comparison between Group-1 and Group-2 patients showing increased TREM-1 expression in the cells of Group-1 revealing the severity of inflammation. <i>n = 8 for both the groups</i>, <i>* p<0</i>.<i>05; NS–Not significant</i></p

Mean fluorescence intensity for the evaluation of gene expression analysis by immunofluorescence using ImageJ software.

<p>The expression levels of CD68, TREM-1, HMGB1 and RAGE were significantly higher in the tissues of Group-1 when compared to Group-2. TREM-2 expression was enhanced in Group-1 but was not significant. The expression of TM and SLX between both the groups was mostly similar and not significant. <i>Group-1 (n = 4)</i>, <i>Group-1 (n = 11) * p<0</i>.<i>05; NS–Not significant</i>.</p

Primers and their sequences used for RT-PCR analysis.

<p>Primers and their sequences used for RT-PCR analysis.</p

The mRNA expression of TREM-1 and TREM-2 genes in tendon tissue.

<p>The expression level of TREM-1 was significantly higher in Group-1 when compared to Group-2 as the control. There was no significant difference in TREM-2 expression between the Group 1 and Group 2. <i>Group-1 (n = 4)</i>, <i>Group-1 (n = 8) * p<0</i>.<i>05; NS–Not significant</i>.</p

MicroRNAs Associated with Shoulder Tendon Matrisome Disorganization in Glenohumeral Arthritis

<div><p>The extracellular matrix (ECM) provides core support which is essential for the cell and tissue architectural development. The role of ECM in many pathological conditions has been well established and ECM-related abnormalities leading to serious consequences have been identified. Though much has been explored in regards to the role of ECM in soft tissue associated pathologies, very little is known about its role in inflammatory disorders in tendon. In this study, we performed microRNA (miRNA) expression analysis in the long head of the human shoulder biceps tendon to identify key genes whose expression was altered during inflammation in patients with glenohumeral arthritis. We identified differential regulation of matrix metalloproteinases (MMPs) that could be critical in collagen type replacement during tendinopathy. The miRNA profiling showed consistent results between the groups and revealed significant changes in the expression of seven different miRNAs in the inflamed tendons. Interestingly, all of these seven miRNAs were previously reported to have either a direct or indirect role in regulating the ECM organization in other pathological disorders. In addition, these miRNAs were also found to alter the expression levels of MMPs, which are the key matrix degrading enzymes associated with ECM-related abnormalities and pathologies. To our knowledge, this is the first report which identifies specific miRNAs associated with inflammation and the matrix reorganization in the tendons. Furthermore, the findings also support the potential role of these miRNAs in altering the collagen type ratio in the tendons during inflammation which is accompanied with differential expression of MMPs.</p></div

Genes associated with COL1A2, COL3A1, MMP9 and MMP2 as determined by NetworkAnalyst.

<p>The NetworkAnalyst program data reveals the genes interconnected with our genes of interest (COL1A2, COL3A1, MMP9 and MMP2) based on the published data base and the gene symbols are based on official gene symbol.</p

NFR staining in shoulder tendon showing the morphology and orientation.

<p>(A) Group 1 patient (representative of 4 individual subjects in Group 1) with tendinopathy as well as glenohumeral arthritis and (B) Group 2 patient (representative of 4 individual subjects in Group 2) with tendinopathy but without glenohumeral arthritis. The green arrows show tendon cells, red arrows point blood vessels indicating angiogenesis, blue arrows indicate ECM disorganization, black arrows show inflammation and yellow arrow mark normal ECM. The figures were taken in 400x magnification.</p

Highly altered miRNAs associated with the genes of interest (COL1A2, COL3A1, MMP9 and MMP2) as determined by miRNA array of Group 1 (n = 4) vs Group 2 (n = 4) of shoulder tendons.

<p>The table displays miRNAs with 10 or more target genes from the microarray data of 1001 miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168077#pone.0168077.s002" target="_blank">S2 Table</a>).</p

Movat pentachrome staining of shoulder biceps tendons.

<p>(A) Group 1 patient (representative of 4 individual subjects in Group 1) with tendinopathy as well as glenohumeral arthritis and (B) Group 2 patient (representative of 4 individual subjects in Group 2) with tendinopathy but without glenohumeral arthritis. The red arrows indicate fibrosis and muscle fibers, yellow arrows show collagen fibers, green arrows mark tenocytes and black arrows show mucin deposits.</p