Background
Rotator cuff tear (RCT) is one of the most common musculoskeletal disorder in orthopedics department. Earlier in 1991, the prevalence of shoulder disorders in people aged 70 and above was 21% [
1]. According to a survey performed by Hiroshi in 2012, the prevalence of RCT was 22.1% was in the general population, and the prevalence was significantly increased with advanced age [
2]. The disease is characterized as injury of one or more tendons/
muscles in
rotator cuff, which can cause pain, stiffness, muscle weakness, and functional limitation. Common risk factors for RCT include advancing age, overuse of shoulder, trauma, and shoulder arthritis [
3].
Surgery is an effective solution for patients with large and full-thickness RCT. Currently, surgical procedures include mini-open and all-arthroscopic rotator cuff repair. According to the follow-up results, there is no significant difference in clinical prognosis between the two methods [
4]. However, the failure rate was reported ranged from 23 to 77% in arthroscopically repaired massive rotator cuff tears, which characterized as the size of tear was more than 5 cm or the tear involving more than two cuff tendons [
5‐
7]. The size of the defect, tendon retraction, muscular atrophy, and degree of fatty degeneration were regarded as failure elements and resulted in higher rate of no-heal and re-tear [
8,
9]. Fatty infiltration was introduced and staged by Goutallier et al. in 1994 and was viewed as a key factor responsible for the higher re-tear rates and poorer functional prognosis [
10,
11]. Fatty degeneration of the infraspinatus was reported as the most independent predictor on outcomes following rotator cuff repair [
12]. Chung et al. also found fatty infiltration of the infraspinatus is a significantly positive correlated factor with healing failure rate after arthroscopic repair of a massive rotator cuff tear [
6]. Another cohort study performed by Gladstone et al. demonstrated that both muscle atrophy and fatty infiltration of the rotator cuff muscles cast significant impacts on regulating cuff repair and functional recovery [
13]. Therefore, an increasing large number of research groups are focusing on the specific mechanism of fatty infiltration.
Till now, the key genes and pathway involved in the fatty infiltration of rotator cuff tear still unclear. Ren et al. identified differentially expressed genes and signaling pathways involved in rotator cuff tear through bioinformatics analysis [
14]. Their study focused on the different genes between torn tendon samples and intact samples. In the present study, to explore differential gene expression of the differential stage of RCT, we examined one published gene dataset available in the Gene Expression Omnibus (GEO) database. Differential expression of genes during each developmental stages of massive rotator cuff tear was examined by comprehensive bioinformatics analyses. Then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, and a protein–protein interaction (PPI) network was used to screen for crucial genes and pathways in the pathophysiological processes of fatty infiltration. Chief sub-PPI networks are explored by MCODE.
Discussion
The mechanism of fatty infiltration of the rotator cuff is complex and still unclear. Using a mouse model constructed by unilateral supraspinatus and infraspinatus tendon tear, Wang et al. demonstrated atrophy and fatty infiltration of muscle exist in the 6-week delayed repair group. And the severity of fatty infiltration was closely linked with the shoulder function [
20]. Another massive rotator cuff tear model caused by a full-thickness tenectomy of the right supraspinatus and infraspinatus in rats showed that the muscle fiber force is decreased and expressions of fibrosis and lipid accumulation related genes were increased [
21]. A further study performed by Liu et al. demonstrated that the main cellular sources involved in fatty infiltration were Tie2 + progenitor cells and fibro/adipogenic progenitor cells [
22]. These three studies were consistent with the clinical reports and researches. In a prospective study aimed to massive rotator cuff tears patients younger than 65 years, 67.9% were demonstrated with a stage 3 or 4 fatty infiltration of the supraspinatus muscle and 47.3% were suffered from a stage 3 or 4 fatty infiltration of the infraspinatus muscle [
23]. And patients with a high degree of fatty infiltration exhibited poorer prognosis after surgery. Patients with grade 3 fatty degeneration showed significant improvement after arthroscopic rotator cuff repair compared to those patients who graded as Goutallier stage IV after nearly 3 years of follow-up [
24]. However, a successful repair of rotator cuff would not bring a significant improvement of fatty infiltration and atrophy of the rotator cuff [
13]. So, studies focused on the cellular and molecular basis of fatty infiltration are of great significance in the prevention and treatment of fatty infiltration in rotator cuff tear.
In the present study, a bioinformatics analysis was used to analyze an available database downloaded from GEO. According to our results, a total of 1089 DEGs were identified as the key mediators in the pathophysiological processes of fatty infiltration. Moreover, we also performed GO annotation and KEGG pathway enrichment analyses to potential biological functions and pathways involved in fatty infiltration. In addition, 259 hub genes including Tmprss11d, Ptprc, Itgam, Mmp9, Tlr2, Il1b, Il18, Ccl5, Cxcl10, and Ccr7 and six modules with important functions were identified from the PPI network.
At present, there are only a few theories and research results about the mechanism of fatty infiltration in rotator cuff. Tmprss11d, a member of type II transmembrane serine protease (TTSP) family, was highly expressed in respiratory epithelium and proved to exert an important role in the host defense system [
25]. Till now, the role of TMPRSS11D in the musculoskeletal system was not clear. Cao et al. reported higher expression of TMPRSS11D is closely linked with the severity of the cancer because of its ability to disintegrate the extracellular matrix [
26]. TMPRSS11D was also demonstrated to have a relationship with fat metabolism in the liver [
25]. Therefore, it is an important research direction of the role of TMPRSS11D in the degeneration of tendon and lipid metabolism in the future. MMP family was also identified as a major player in rotator cuff tear. Protein expressions of MMP-9 and MMP-13 were notably increased in rotator cuff specimens obtained from surgery patients [
27]. However, in MMP-13 (−/−) knockout mice group, researchers found a higher amount of fat infiltration compared to the wildtype MMP-13 (+/+) mice group. These interesting results indicated that MMP-13 exert a significant role in rotator muscle fatty infiltration and may be a potential target for treatment [
28]. MMP inhibitor was helpful in the improvement of tendon-to-bone healing in a rotator cuff repair model [
29]. Moreover, Tlr2, another identified hub gene in our study, was highly expressed in rotator cuff tendon in the first 2 weeks following injuries [
30]. Tlr2 was viewed as an important mediator in NLRP3 inflammasome. The lowered activity of NLRP3 leads to a decrease in the fat accumulation in hepatocytes [
31]. So, the molecular mediators associated with NLRP3 inflammasome was worth studying in the pathology of fatty infiltration in rotator cuff tendons. Taken together, hub genes identified in our research by comprehensive bioinformatic analyses were meaningful and worthy of deep-going study in the future.
We also performed an intensive module analysis based on the PPI network in this study. In chronically rotator cuff muscles, increased gene expressions of PPAR-γ, PLD1, miR-27a and lipid synthesis were obversed. And ECM degradation- and remodeling-related genes such as MMP2, MMP9, and MMP14 were also overexpressed in the pathological process of fibrosis [
32]. Blocking of p38 MAPK signaling, an important regulator of adipogenesis, reduced the fat accumulation by nearly half and adjusted the expressions of adipogenesis- and matrix accumulation-related genes [
33]. Our KEGG pathway enrichment analyses showed that the functions of module 4 and 5 were mainly associated with metabolic pathways and fatty acid degradation. And DEGs such as Acsl1, Acsl4, Acsl6, Lipg, Lpin1, Mboat2, Pnpla3, Ppap2a, Ccbl1, Impdh1, and Nt5c1a were functionality associated with these pathways.
A limitation of this study is that the stage of fatty infiltration in the rat models in GSE103266 is unclear. Besides, there are some anatomical differences between human and rat. Therefore, the pathophysiological process of fatty infiltration in rat is different from human. We still need to conduct validation experiment to proof our speculation in the future.
Conclusion
The current study identified a series of DEGs in each stage of massive rotator cuff tear. Using a series of bioinformatics analyses, hub genes including Tmprss11d, Ptprc, Itgam, Mmp9, Tlr2, Il1b, Il18, Ccl5, Cxcl10, and Ccr7 were identified. Further GO and KEGG pathway analysis suggests potential mechanisms through which these hub genes mediate fatty infiltration pathogenesis in rotator cuff tear. In addition, module analysis highlights the important roles of metabolic pathways and fatty acid degradation in the regulation of fatty infiltration. Our results indicated the potential key genes and pathways involved in fatty degeneration and supplied therapeutic targets to improve clinical outcomes for patients who suffer from chronic rotator cuff tears and fatty infiltration.