Analysis of differentially expressed genes in torn rotator cuff tendon tissues in diabetic patients through RNA-sequencing.

BACKGROUND: Rotator cuff tears (RCT) is a common musculoskeletal disorder in the shoulder which cause pain and functional disability. Diabetes mellitus (DM) is characterized by impaired ability of producing or responding to insulin and has been reported to act as a risk factor of the progression of rotator cuff tendinopathy and tear. Long non-coding RNAs (lncRNAs) are involved in the development of various diseases, but little is known about their potential roles involved in RCT of diabetic patients. METHODS: RNA-Sequencing (RNA-Seq) was used in this study to profile differentially expressed lncRNAs and mRNAs in RCT samples between 3 diabetic and 3 nondiabetic patients. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed to annotate the function of the differentially expressed genes (DEGs). LncRNA-mRNA co-expression network and competing endogenous RNA (ceRNA) network were constructed to elucidate the potential molecular mechanisms of DM affecting RCT. RESULTS: In total, 505 lncRNAs and 388 mRNAs were detected to be differentially expressed in RCT samples between diabetic and nondiabetic patients. GO functional analysis indicated that related lncRNAs and mRNAs were involved in metabolic process, immune system process and others. KEGG pathway analysis indicated that related mRNAs were involved in ferroptosis, PI3K-Akt signaling pathway, Wnt signaling pathway, JAK-STAT signaling pathway and IL-17 signaling pathway and others. LncRNA-mRNA co-expression network was constructed, and ceRNA network showed the interaction of differentially expressed RNAs, comprising 5 lncRNAs, 2 mRNAs, and 142 miRNAs. TF regulation analysis revealed that STAT affected the progression of RCT by regulating the apoptosis pathway in diabetic patients. CONCLUSIONS: We preliminarily dissected the differential expression profile of lncRNAs and mRNAs in torn rotator cuff tendon between diabetic and nondiabetic patients. And the bioinformatic analysis suggested some important RNAs and signaling pathways regarding inflammation and apoptosis were involved in diabetic RCT. Our findings offer a new perspective on the association between DM and progression of RCT.

Gene Expression in Synovium of Rotator Cuff Tear Patients Determined by RNA Sequencing.

Rotator cuff tear (RCT) is a common shoulder disorder related to pain and dysfunction. However, the pathological mechanism of RCT remains unclear. Thus, this study aims to investigate the molecular events in RCT synovium and identify possible target genes and pathways as determined by RNA sequencing (RNA-Seq). The synovial tissue was biopsied from 3 patients with RCT (RCT group) and 3 patients with shoulder instability (Control group) during arthroscopic surgery. Then, differentially expressed (DE) mRNAs, long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs) were comprehensively profiled by RNA-Seq. Gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and competing endogenous RNA (ceRNA) network analysis were performed to identify the potential functions of these DE genes. 447 mRNAs, 103 lncRNAs and 15 miRNAs were identified differentially expressed. The DE mRNAs were highlighted in inflammatory pathway including up-regulated T cell costimulation, positive regulation of T cell activation, and T cell receptor signaling. Down-regulated fatty acid degradation pathway and 5'-AMP-activated protein kinase (AMPK) signaling in RCT group are also enriched. Validation assay showed that the expression of pro-inflammatory molecules including IL21R, CCR5, TNFSF11, and MMP11 was significantly increased in RCT group compared with Control group. CeRNA analysis further revealed lncRNA-miRNA-mRNA regulatory networks involving IL21R and TNFSF11 in RCT. Activated synovial inflammation is the remarkable event of RCT. Importantly, increased T cell activation and disordered fatty acid metabolism signaling might play a significant role. ceRNA networks involving IL21R and TNFSF11 identified could potentially control the progression of RCT. In conclusion, our findings could provide new evidence for the molecular mechanisms of RCT and might identify new therapeutic targets.

Factors associated with genetic markers for rotator cuff disease in patients with atraumatic rotator cuff tears.

For atraumatic rotator cuff tears, genetics contributes to symptomatic tear risk and may influence rotator cuff healing after surgical repair. But little is known about how genetic factors influence rotator cuff tear patient characteristics at presentation. We collected saliva samples for genotyping from atraumatic rotator cuff tear patients. We examined nine single nucleotide polymorphisms (SNPs) associated with cuff tears in prior literature. We estimated associations of SNP dosage with (1) age at tear diagnosis, (2) bilateral atraumatic tear prevalence, and (3) tear size. Linear regression was used to estimate associations with diagnosis age adjusted for sex and principal components. Logistic regression and ordinal logistic regression were used to estimate associations with bilateral tear prevalence and tear size category, respectively, adjusting for age, sex, and principal components. Of 344 eligible patients, 336 provided sufficient samples for genotyping. Median age at tear diagnosis was 61, 22% (N = 74) had bilateral atraumatic tears, and 9% (N = 29) had massive tears. SNP rs13107325 in the SLC39A8 gene and rs11850957 in the STXBP6 gene were associated with younger diagnosis age even after accounting for multiple comparisons (rs13107325: -4 years, 95% CI = -6.5, -1.4; rs11850957: -2.7 years, 95% CI = -4.3, -1.1). No other significant associations were observed with diagnosis age, tear size, or bilateral tear prevalence. SLC39A8 encodes a Mn transporter. STXBP6 may play a role in inflammatory responses by altering phagocytosis and antigen presentation of monocytes and macrophages. Further research is needed to determine if genetic markers can be used alongside patient characteristics to aid in identifying optimal surgical repair candidates.

Influence of Vitamin D Deficiency on the Expression of Genes and Proteins in Patients With Medium Rotator Cuff Tears.

BACKGROUND: Whether vitamin D deficiency is related to rotator cuff muscle and tendon physiology is controversial. PURPOSE: To assess the relationship between vitamin D deficiency and various gene expression patterns in patients with rotator cuff tears. STUDY DESIGN: Controlled laboratory study. METHODS: During arthroscopic surgery, samples from the supraspinatus muscle, deltoid muscle, and supraspinatus tendon were acquired from 12 patients with vitamin D deficiency (serum 25-hydroxyvitamin D concentration <20 ng/dL) and 12 patients with sufficient vitamin D levels (control group, serum 25-hydroxyvitamin D concentration ≥30 ng/dL) who were matched for age, sex, and tear size. Alterations in the expression of genes and proteins associated with myogenesis, muscle atrophy, adipogenesis, inflammation, and apoptosis, as well as in vitamin D receptor expression, were assessed using quantitative reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry and were compared between the 2 groups. RESULTS: Vitamin D receptor gene expression in the deltoid muscle was significantly lower in the vitamin D deficiency group than in the control group (P = .043). Additionally, in the deltoid muscle, myoDgene expression levels were lower and atrogin levels were higher in the vitamin D deficiency group than in the control group (P = .034 and P = .011, respectively). However, in the supraspinatus muscle, no differences were observed between groups in the expression of myogenesis- or atrophy-related genes (all P > .05). The expression of inflammation-related genes (interleukin (IL)-1ß and IL-6) was significantly higher in the vitamin D deficiency group, in both the deltoid and supraspinatus muscles (all P < .05). The supraspinatus tendon tissue did not show any significant differences in any gene expression evaluated (all P > .05). A correlation between gene and protein expression was observed for atrogin and IL-1ß in the deltoid muscle (P = .019 and P = .037, respectively) and for IL-6 in the supraspinatus muscle (P = .044). CONCLUSION: Vitamin D deficiency was not associated with the expression of myogenesis-related or muscle atrophy-related genes in the supraspinatus muscle of patients with rotator cuff tears, unlike in the deltoid muscle; rather, vitamin D deficiency was associated with increased proinflammatory cytokine expression. CLINICAL RELEVANCE: In patients with rotator cuff tears, vitamin D deficiency was observed to be associated with increased levels of proinflammatory cytokines in the rotator cuff muscles, without significant changes in gene expression related to myogenesis or muscle atrophy.

Mechanisms of ferroptosis with immune infiltration and inflammatory response in rotator cuff injury.

The processes driving ferroptosis and rotator cuff (RC) inflammation are yet unknown. The mechanism of ferroptosis and inflammation involved in the development of RC tears was investigated. The Gene Expression Omnibus database was used to obtain the microarray data relevant to the RC tears for further investigation. In this study, we created an RC tears rat model for in vivo experimental validation. For the additional function enrichment analysis, 10 hub ferroptosis-related genes were chosen to construct the correlation regulation network. In RC tears, it was discovered that genes related to hub ferroptosis and hub inflammatory response were strongly correlated. The outcomes of in vivo tests showed that RC tears were related to Cd68-Cxcl13, Acsl4-Sat1, Acsl3-Eno3, Acsl3-Ccr7, and Ccr7-Eno3 pairings in regulating ferroptosis and inflammatory response. Thus, our results show an association between ferroptosis and inflammation, providing a new avenue to explore the clinical treatment of RC tears.

Association between the rs820218 Variant within the SAP30BP Gene and Rotator Cuff Rupture in an Amazonian Population.

BACKGROUND: Rotator cuff disease is one of the leading causes of musculoskeletal pain and disability, and its etiology is most likely multifactorial but remains incompletely understood. Therefore, the objective of this research was to investigate the relationship of the single-nucleotide rs820218 polymorphism of the SAP30-binding protein (SAP30BP) gene with rotator cuff tears in the Amazonian population. METHODS: The case group consisted of patients who were operated on due to rotator cuff tears in a hospital in the Amazon region between 2010 and 2021, and the control group was composed of individuals who were selected after negative physical examinations for rotator cuff tears. Genomic DNA was obtained from saliva samples. For the genotyping and allelic discrimination of the selected single nucleotide polymorphism (rs820218) in the SAP30BP gene, real-time PCR was performed. RESULTS: The frequency of the A allele in the control group was four times as high as that in the case group (AA homozygotes); an association of the genetic variant rs820218 of the SAP30BP gene with rotator cuff tears was not established (p = 0.28 and 0.20), as the A allelic frequency is ordinarily low in the general population. CONCLUSIONS: The presence of the A allele indicates protection against rotator cuff tears.

Pathological alterations in the expression status of rotator cuff tendon matrix components in hyperlipidemia.

Hyperlipidemia is an important risk factor in the development and progression of tendon pathology, however its role in aggravating rotator cuff tendon injury (RCTI) is largely unknown. We aimed to assess the expression status of key extracellular matrix (ECM) components in the tendon tissues and tenocytes under hyperlipidemia. Shoulder rotator cuff (RC) tendon tissues harvested from the swine model of hyperlipidemia displayed alterations in histomorphometry and the expression status of major ECM component proteins including COL-I, COL-III, COL-IV, COL-V, COL-VI, MMP2, and MMP9. Similarly, the LDL- and oxLDL-challenged tenocytes displayed altered expression of the same proteins at both transcriptional and translational levels. In addition, the lipid uptake and cellular reactive oxygen radicals predominated in the lipid-challenged tenocytes compared to the control. Overall, the LDL-treated cells displayed predominant pathological alterations compared to the ox-LDL-treated cells. Further understanding regarding the underlying molecular mechanisms driving the tendon matrisome alteration and subsequent aggravated RCTI pathology in hyperlipidemia could open novel translational avenues in the management of RCTI.

mRNA and long non-coding RNA expression profiles of rotator cuff tear patients reveal inflammatory features in long head of biceps tendon.

BACKGROUND: This study aimed to identify the differentially expressed mRNAs and lncRNAs in inflammatory long head of biceps tendon (LHBT) of rotator cuff tear (RCT) patients and further explore the function and potential targets of differentially expressed lncRNAs in biceps tendon pathology. METHODS: Human gene expression microarray was made between 3 inflammatory LHBT samples and 3 normal LHBT samples from RCT patients. GO analysis and KEGG pathway analysis were performed to annotate the function of differentially expressed mRNAs. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was admitted to verify their expression. LncRNA-mRNA co-expression network, cis-acting element, trans-acting element and transcription factor (TF) regulation analysis were constructed to predict the potential molecular regulatory mechanisms and targets for LHB tendinitis. RESULTS: 103 differentially expressed lncRNAs and mRNAs, of which 75 were up-regulated and 28 were down-regulated, were detected to be differentially expressed in LHBT. The expressions of 4 most differentially expressed lncRNAs (A2MP1, LOC100996671, COL6A4P, lnc-LRCH1-5) were confirmed by qRT-PCR. GO functional analysis indicated that related lncRNAs and mRNAs were involved in the biological processes of regulation of innate immune response, neutrophil chemotaxis, interleukin-1 cell response and others. KEGG pathway analysis indicated that related lncRNAs and mRNAs were involved in MAPK signaling pathway, NF-kappa B signaling pathway, cAMP signaling pathway and others. TF regulation analysis revealed that COL6A4P2, A2MP1 and LOC100996671 target NFKB2. CONCLUSIONS: LlncRNA-COL6A4P2, A2MP1 and LOC100996671 may regulate the inflammation of LHBT in RCT patients through NFKB2/NF-kappa B signaling pathway, and preliminarily revealed the pathological molecular mechanism of tendinitis of LHBT.

Mechanical stimulation promotes enthesis injury repair by mobilizing Prrx1+ cells via ciliary TGF-ß signaling.

Proper mechanical stimulation can improve rotator cuff enthesis injury repair. However, the underlying mechanism of mechanical stimulation promoting injury repair is still unknown. In this study, we found that Prrx1+ cell was essential for murine rotator cuff enthesis development identified by single-cell RNA sequence and involved in the injury repair. Proper mechanical stimulation could promote the migration of Prrx1+ cells to enhance enthesis injury repair. Meantime, TGF-ß signaling and primary cilia played an essential role in mediating mechanical stimulation signaling transmission. Proper mechanical stimulation enhanced the release of active TGF-ß1 to promote migration of Prrx1+ cells. Inhibition of TGF-ß signaling eliminated the stimulatory effect of mechanical stimulation on Prrx1+ cell migration and enthesis injury repair. In addition, knockdown of Pallidin to inhibit TGF-ßR2 translocation to the primary cilia or deletion of Ift88 in Prrx1+ cells also restrained the mechanics-induced Prrx1+ cells migration. These findings suggested that mechanical stimulation could increase the release of active TGF-ß1 and enhance the mobilization of Prrx1+ cells to promote enthesis injury repair via ciliary TGF-ß signaling.

RNA-Seq analysis reveals sex-dependent transcriptomic profiles of human subacromial bursa stratified by tear etiology.

Rotator cuff tendinopathy, a major cause of shoulder disability, occurs due to trauma or degeneration. Our molecular understanding of traumatic and degenerative tears remains elusive. Here, we probed transcript level differences between traumatic and degenerative tears. Subacromial bursa tissues were collected from patients with traumatic or degenerative tears during arthroscopy (N = 32). Transcripts differentially expressed by tear etiology were detected by RNA-seq. RNA-seq results were validated by real-time quantitative polymerase chain reaction. We identified 334 protein-coding transcripts differentially expressed between traumatic and degenerative tears in females and 167 in males at a fold-change greater than 2. In females, XIRP2, MYL1, MYBPC1, TNNT1, and LMOD2, were highly expressed in traumatic tears whereas TPSD1, CDSN, RCVRN, LTBP4, and PTGS1 were elevated in degen tears. Transcripts elevated in traumatic tears represented muscle cell differentiation and development, and muscle contraction whereas those elevated in degenerative tears represented cell activation and immune response. In males, AZGP1, CNTFR, COL9A1, ZNF98, and EREG were highly elevated in traumatic tears whereas MYL2, HOXD11, SLC6A7, CADM1, and MMP17 were highly expressed in degenerative tears. Transcripts elevated in traumatic tears represented metabolic/catabolic processes, and transmembrane protein transport while processes related to cell cycle were mainly enriched in degenerative tears. Numerous long noncoding RNAs were differentially expressed between traumatic and degenerative tears in both sexes. In summary, this study provides insights into molecular biology of bursa in patients with rotator cuff tendon disease based on tear acuity and novel sex-based transcript differences that could inform clinical decision making in treating patients with traumatic or degenerative shoulder injuries.

A Hyperglycemic Microenvironment Inhibits Tendon-to-Bone Healing through the let-7b-5p/CFTR Pathway.

BACKGROUND: Tendon-to-bone healing is a difficult process in treatment of rotator cuff tear (RCT). In addition, diabetes is an important risk factor for poor tendon-to-bone healing. Therefore, we investigated the specific mechanisms through which diabetes affects tendon-to-bone healing by regulating the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). METHODS: Tendon-derived stem cells (TDSCs) were extracted from rats after which their proliferative capacities were evaluated by the MTT assay. The expression levels of CFTR and tendon-related markers were determined by qRT-PCR. Then, bioinformatics analyses and dual luciferase reporter gene assays were used to identify miRNAs with the ability to bind CFTR mRNA. Finally, CFTR was overexpressed in TDSCs to validate the specific mechanisms through which the high glucose microenvironment inhibits tendon-to-bone healing. RESULTS: The high glucose microenvironment downregulated mRNA expression levels of tendon-related markers and CFTR in TDSCs cultured with different glucose concentrations. Additionally, bioinformatics analyses revealed that let-7b-5p may be regulated by the high glucose microenvironment and can regulate CFTR levels. Moreover, a dual luciferase reporter gene assay was used to confirm that let-7b-5p targets and binds CFTR mRNA. Additional experiments also confirmed that overexpressed CFTR effectively reversed the negative effects of the hyperglycaemic microenvironment and upregulation of let-7b-5p on TDSC proliferation and differentiation. These findings imply that the hyperglycemic microenvironment inhibits CFTR transcription and, consequently, proliferation and differentiation of TDSCs in vitro by upregulating let-7b-5p. CONCLUSIONS: A hyperglycemic microenvironment inhibits TDSC proliferation in vitro via the let-7b-5p/CFTR pathway, and this is a potential mechanism in diabetes-induced poor tendon-to-bone healing.

RNA-Seq reveals distinct transcriptomic differences in rotator cuff tendon based on tear etiology and patient sex.

Rotator cuff tears are a common pathology in the shoulder and generally have two underlying etiologies: traumatic and degenerative. Little is known about the molecular underpinning of these etiologies. Here we queried transcript level differences in tear etiology stratified by sex in 31 patients with rotator cuff tears. Tendon tissues were isolated from females (N = 16) and males (N = 15) with traumatic (N = 16) or degenerative (N = 15) tears during arthroscopy. Differentially expressed transcripts were identified by RNA-seq and biological processes were probed computationally. Expression of some transcripts was validated by real-time quantitative polymerase chain reaction (qPCR). We identified 339 and 336 transcripts differentially expressed by tear etiology in females and males, respectively, at a fold-change greater than |2|. In females, GSTM1, MT1G, S1008A, ACSM3, DSC, FAM110C, and VNN2 were elevated in traumatic tears representing metabolic/catabolic processes, and immune response whereas CHAD, CLEC3A, IBSP, TNMD, APLNR, and CPA3 were elevated in degenerative tears representing tissue morphogenesis and developmental processes, angiogenesis, and extracellular matrix organization. In males, ELOA3B, CXCL8, ADM, TNS4, and SPOCK1 were elevated in traumatic tears representing localization of endoplasmic reticulum, chromosome organization, leukocyte/neutrophil degranulation, and protein transport whereas MYL2, TNNC1, MB, CPA3, APLNR, and CA3 were highly upregulated in degenerative tears representing muscle cell differentiation and development and angiogenesis. Numerous novel lncRNAs were identified to be differentially expressed by tear etiology in both sexes. Real-time qPCR confirmed RNA-seq data. This study improves our understanding of tendon biology based on underlying etiology (trauma or degeneration) in a sex-specific manner. These findings may help drive clinical decision-making in females and males with traumatic and degenerative shoulder injuries.

Inhibition of miR-205 promotes proliferation, migration and fibrosis of tenocytes through targeting MECP2: Implications for rotator cuff injury.

BACKGROUND: The production of inflammatory mediators is critical for tenocytes proliferation and migration, which play an important role in rotator cuff injury repair and regulation of collagen. MicroRNA (miRNA)-205 (miR-205) promotes the secretion of inflammatory factors. The mechanism of the tenocytes regulation by miR-205 remains unknown. In this paper, we showed that miR-205 can regulate the proliferation, migration and fibrosis of tenocytes. OBJECTIVES: To investigate the function and mechanism of miR-205/MeCP2 pathway on the proliferation, migration and fibrosis of rotator cuff tenocytes, in order to provide a new perspective on the repair of rotator cuff tear injury. MATERIAL AND METHODS: The tenocytes were collected under sterile conditions from the Achilles tendons of Sprague Dawley (SD) rats (weighing 150-200 g). The cells of passages 2-4 were used for the following experiments. All miRNA and vectors were transfected with Lipofectamine 2000. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR), Cell Counting Kit-8 (CCK-8) assay, luciferase reporter assay, and migration assay were performed. Then, immunoblotting analysis and statistical analysis were conducted. RESULTS: The CCK-8 and migration assay revealed that miR-205 inhibition resulted in increased tenocytes proliferation, migration and fibrosis. The miR-205 reduced the mRNA and protein expression levels of MECP2, which is involved in cell proliferation and migration of tenocytes. The miR-205 inhibited luciferase intensity under the control of the 3'UTRs of MECP2. CONCLUSIONS: The inhibition of MECP2 reversed the effect of miR-205 inhibitor on tenocytes, including the proliferation and migration of tenocytes, indicating that miR-205 may be valuable in miRNA-based therapies for rotator cuff injury.

Rotator Cuff Tear Size Regulates Fibroadipogenic Progenitor Number and Gene Expression Profile in the Supraspinatus Independent of Patient Age.

BACKGROUND: Fatty infiltration of rotator cuff muscle is a limiting factor in the success of repairs. Fibroadipogenic progenitors (FAPs) are a population of stem cells within the rotator cuff that can differentiate into white adipocytes, fibroblasts, and beige adipocytes. The effects of patient age and rotator cuff tendon tear size on the number, differentiation patterns, and gene expression profiles of FAPs have not yet been analyzed. PURPOSE: To determine if patient age and rotator cuff tear size independently regulate FAP number, differentiation patterns, and gene expression profiles. STUDY DESIGN: Controlled laboratory study. METHODS: Supraspinatus muscle samples were collected from 26 patients between the ages of 42 and 76 years with partial- or full-thickness rotator cuff tears. FAPs were quantified using fluorescence-activated cell sorting. Gene expression analysis was performed across a custom 96-gene panel using NanoString. In vitro differentiation assays of FAPs were conducted using adipogenic, fibrogenic, and beige-inducing (amibegron-treated) media, and quantitative polymerase chain reaction was used to assess gene expression differences between adipogenic and amibegron media conditions. Multivariable linear regressions were performed using Stata to independently analyze the effects of age and rotator cuff tear size on FAP number, differentiation, and gene expression. RESULTS: Increasing age and tear size were independently correlated with increased FAP number (ßage = 0.21, P = .03; ßtear size = 3.86, P = .05). There was no clear association between age and gene expression of freshly sorted FAPs. Under adipogenic and fibrogenic media conditions, increasing age and tear size were independently associated with increased adipogenic and fibrogenic differentiation of FAPs. Under amibegron treatment conditions, age positively correlated with increased beige differentiation (ß = 1.03; P < .0001), while increasing tear size showed a trend toward decreased beige differentiation (ß = -4.87; P = .1). When gene expression patterns between adipogenic and amibegron media conditions were compared, larger tear size strongly inhibited beige gene expression, while advanced age did not. CONCLUSION: Patient age and rotator cuff tear size independently regulated FAP number, differentiation, and gene expression. Age and tear size were positively correlated with increased FAP number and fibrogenic/adipogenic differentiation. Advancing patient age did not limit FAP beige differentiation and gene expression, while increasing rotator cuff tear size strongly inhibited these processes.

Histological and molecular features of the subacromial bursa of rotator cuff tears compared to non-tendon defects: a pilot study.

BACKGROUND: The role of the subacromial bursa in the development or healing of shoulder pathologies is unclear. Due to this limited knowledge, we aimed to understand specific reactions of the subacromial bursa according to rotator cuff (RC) pathologies compared to non-tendon defects of the shoulder. We hypothesized that the tissue composition and inflammatory status of the bursa are likely to vary between shoulder pathologies depending on the presence and the extent of RC lesion. METHOD: Bursa samples from patients with either 1) shoulder instability with intact RC (healthy bursa, control), 2) osteochondral pathology with intact RC, 3) partial supraspinatus (SSP) tendon tear, or 4) full-thickness SSP tear were investigated histologically and on gene expression level. RESULT: Bursae from SSP tears differed from non-tendon pathologies by exhibiting increased chondral metaplasia and TGFß1 expression. MMP1 was not expressed in healthy bursa controls, but strongly increased with full-thickness SSP tears. Additionally, the expression of the inflammatory mediators IL1ß, IL6, and COX2 increased with the extent of SSP tear as shown by correlation analysis. In contrast, increased angiogenesis and nerve fibers as well as significantly upregulated IL6 and COX2 expression were features of bursae from patients with osteochondral pathology. Using immunohistochemistry, CD45+ leukocytes were observed in all examined groups, which were identified in particular as CD68+ monocytes/macrophages. CONCLUSION: In summary, besides the strong increase in MMP1 expression with SSP tear, molecular changes were minor between the investigated groups. However, expression of pro-inflammatory cytokines correlated with the severity of the SSP tear. Most pronounced tissue alterations occurred for the osteochondral pathology and full-thickness SSP tear group, which demonstrates that the bursal reaction is not exclusively dependent on the occurrence of an SSP tear rather than longstanding degenerative changes. The present bursa characterization contributes to the understanding of specific tissue alterations related to RC tears or non-tendon shoulder pathologies. This pilot study provides the basis for future studies elucidating the role of the subacromial bursa in the development or healing of shoulder pathologies.

Increasing transforming growth factor-beta concentrations with age decrease apelin in the rat rotator cuff.

BACKGROUND: The rotator cuff undergoes natural degeneration with age, leading to age-related rotator cuff tear; however, the precise mechanism remains unclear. Transforming growth factor-beta (TGF-ß) concentrations rise with age and TGF-ß contributes to the pathophysiology of skeletal muscle. TGF-ß has also been shown to suppress expression of the myokine, apelin, in skin fibroblasts. We hypothesized that TGF-ß expression in the rotator cuff changes with age and regulates apelin expression, thereby contributing to rotator cuff degeneration. METHODS: We used quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) to measure the expression of apelin and tendon-related genes (Tnmd, Col1a1, and Col3a1) in the rotator cuff of young (12 weeks), adult (24 weeks), and old (48 weeks) rats. Using Q-RT-PCR and enzyme-linked immunosorbent assay, we also measured Tgfb mRNA and TGF-ß protein levels, respectively. Furthermore, we used Q-RT-PCR to measure apelin mRNA levels in rotator cuff-derived cells after treatment with 0 (control) and 10 ng/mL recombinant TGF-ß. RESULTS: Apelin mRNA levels were significantly lower in old compared to young and adult rats. Similarly, tendon-related genes, Tnmd, Col1a1, and Col3a1, were significantly lower in adult and old rats than young rats. In contrast, Tgfb mRNA and TGF-ß protein were significantly higher in old compared to young rats. Stimulation with exogenous TGF-ß significantly decreased Apelin mRNA expression compared to control. CONCLUSIONS: TGF-ß regulates apelin expression in the rotator cuff and may play a key role in the degenerative pathology of the rotator cuff with age.

Distinct Gene Expression Profile in Patients With Poor Postoperative Outcomes After Rotator Cuff Repair: A Case-Control Study.

BACKGROUND: Impaired healing after rotator cuff repair is a major concern, with retear rates as high as 94%. A method to predict whether patients are likely to experience poor surgical outcomes would change clinical practice. While various patient factors, such as age and tear size, have been linked to poor functional outcomes, it is currently very challenging to predict outcomes before surgery. PURPOSE: To evaluate gene expression differences in tissue collected during surgery between patients who ultimately went on to have good outcomes and those who experienced a retear, in an effort to determine if surgical outcomes can be predicted. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Rotator cuff tissue was collected at the time of surgery from 140 patients. Patients were tracked for a minimum of 6 months to identify those with good or poor outcomes, using clinical functional scores and follow-up magnetic resonance imaging to confirm failure to heal or retear. Gene expression differences between 8 patients with poor outcomes and 28 patients with good outcomes were assessed using a multiplex gene expression analysis via NanoString and a custom-curated panel of 145 genes related to various stages of rotator cuff healing. RESULTS: Although significant differences in the expression of individual genes were not observed, gene set enrichment analysis highlighted major differences in gene sets. Patients who had poor healing outcomes showed greater expression of gene sets related to extracellular matrix production (P < .0001) and cellular biosynthetic pathways (P < .001), while patients who had good healing outcomes showed greater expression of genes associated with the proinflammatory (M1) macrophage phenotype (P < .05). CONCLUSION: These results suggest that a more proinflammatory, fibrotic environment before repair may play a role in poor healing outcome. With validation in a larger cohort, these results may ultimately lead to diagnostic methods to preoperatively predict those at risk for poor surgical outcomes.

Identification of a Novel Genetic Marker for Risk of Degenerative Rotator Cuff Disease Surgery in the UK Biobank.

BACKGROUND: While evidence indicates that familial predisposition influences the risk of developing degenerative rotator cuff disease (RCD), knowledge of specific genetic markers is limited. We conducted a genome-wide association study of RCD surgery using the UK Biobank, a prospective cohort of 500,000 people (40 to 69 years of age at enrollment) with genotype data. METHODS: Cases with surgery for degenerative RCD were identified using linked hospital records. The cases were defined as an International Classification of Diseases, Tenth Revision (ICD-10) code of M75.1 determined by a trauma/orthopaedic specialist and surgery consistent with RCD treatment. Cases were excluded if a diagnosis of traumatic injury had been made during the same hospital visit. For each case, up to 5 controls matched by age, sex, and follow-up time were chosen from the UK Biobank. Analyses were limited to European-ancestry individuals who were not third-degree or closer relations. We used logistic regression to test for genetic association of 674,405 typed and >10 million imputed markers, after adjusting for age, sex, population principal components, and follow-up. RESULTS: We identified 2,917 RCD surgery cases and 14,158 matched controls. We observed 1 genome-wide significant signal (p < 5 × 10-8) for a novel locus tagged by rs2237352 in the CREB5 gene on chromosome 7 (odds ratio [OR] = 1.17, 95% confidence interval [CI] = 1.11 to 1.24). The single-nucleotide polymorphism (SNP) rs2237352 was imputed with a high degree of confidence (info score = 0.9847) and is common, with a minor allele frequency of 47%. After expanding the control sample to include additional unmatched non-cases, rs2237352 and another SNP in the CREB5 gene, rs12700903, were genome-wide significant. We did not detect genome-wide significant signals at loci associated with RCD in previous studies. CONCLUSIONS: We identified a novel association between a variant in the CREB5 gene and RCD surgery. Validation of this finding in studies with imaging data to confirm diagnoses will be an important next step. CLINICAL RELEVANCE: Identification of genetic RCD susceptibility markers can guide understanding of biological processes in rotator cuff degeneration and help inform disease risk in the clinical setting. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

A genome-wide association study for shoulder impingement and rotator cuff disease.

BACKGROUND: The purpose of the study was to identify genetic variants associated with rotator cuff disease by performing a genome-wide association study (GWAS) for shoulder impingement using the UK Biobank (UKB) cohort and then combining the GWAS data with a prior GWAS for rotator cuff tears. The loci identified by the GWAS and meta-analysis were examined for changes in expression following rotator cuff tearing using RNA sequencing. METHODS: A GWAS was performed using data from UKB with 3864 cases of shoulder impingement. The summary statistics from shoulder impingement and a prior study on rotator cuff tears were combined in a meta-analysis. Also, the previous association of 2 single-nucleotide polymorphisms (SNPs) with shoulder impingement from a published GWAS using the UKB was tested. Rotator cuff tendon biopsies were obtained from 24 patients with full-thickness rotator cuff tears who underwent arthroscopic rotator cuff repair (cases) and 9 patients who underwent open reduction internal fixation for a proximal humeral fracture (controls). Total RNA was extracted and differential gene expression was measured by RNA sequencing for genes with variants associated with rotator cuff tearing. RESULTS: The shoulder impingement GWAS identified 4 new loci: LOC100506457, LSP1P3, LOC100506207, and MIS18BP1/LINC00871. Combining data with a prior GWAS for rotator cuff tears in a meta-analysis resulted in the identification of an additional 7 loci: SLC39A8/UBE2D3, C5orf63, ASTN2, STK24, FRMPD4, ACOT9/SAT1, and LINC00890/ALG13. Many of the identified loci have known biologic functions or prior associations with diseases, suggesting possible biologic pathways leading to rotator cuff disease. RNA sequencing experiments show that expression of STK24 increases whereas expression of SAT1 and UBE2D3 decreases following rotator cuff tearing. Two SNPs previously reported to show an association with shoulder impingement from a prior UKB GWAS were not validated in our study. CONCLUSION: This is the first GWAS for shoulder impingement in which new data from UKB enabled the identification of 4 loci showing a genetic association. A meta-analysis with a prior GWAS for rotator cuff tearing identified an additional 7 loci. The known biologic roles of many of the 11 loci suggest plausible biologic mechanisms underlying the etiology of rotator cuff disease. The risk alleles from each of the genetic loci can be used to assess the risk for rotator cuff disease in individual patients, enabling preventative or restorative actions via personalized medicine.