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.

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.

IL-1 reprogramming of adult neural stem cells limits neurocognitive recovery after viral encephalitis by maintaining a proinflammatory state.

Innate immune responses to emerging RNA viruses are increasingly recognized as having significant contributions to neurologic sequelae, especially memory disorders. Using a recovery model of West Nile virus (WNV) encephalitis, we show that, while macrophages deliver the antiviral and anti-neurogenic cytokine IL-1ß during acute infection; viral recovery is associated with continued astrocyte inflammasome-mediated production of inflammatory levels of IL-1ß, which is maintained by hippocampal astrogenesis via IL-1R1 signaling in neural stem cells (NSC). Accordingly, aberrant astrogenesis is prevented in the absence of IL-1 signaling in NSC, indicating that only newly generated astrocytes exert neurotoxic effects, preventing synapse repair and promoting spatial learning deficits. Ex vivo evaluation of IL-1ß-treated adult hippocampal NSC revealed the upregulation of developmental differentiation pathways that derail adult neurogenesis in favor of astrogenesis, following viral infection. We conclude that NSC-specific IL-1 signaling within the hippocampus during viral encephalitis prevents synapse recovery and promotes spatial learning defects via altered fates of NSC progeny that maintain inflammation.

Periostin loss-of-function protects mice from post-traumatic and age-related osteoarthritis.

BACKGROUND: Elevated levels of periostin (Postn) in the cartilage and bone are associated with osteoarthritis (OA). However, it remains unknown whether Postn loss-of-function can delay or prevent the development of OA. In this study, we sought to better understand the role of Postn in OA development and assessed the functional impact of Postn deficiency on post-traumatic and age-related OA in mice. METHODS: The effects of Postn deficiency were studied in two murine experimental OA models using Postn-/- (n = 32) and littermate wild-type (wt) mice (n = 36). Post-traumatic OA was induced by destabilization of the medial meniscus (DMM) in 10-week-old mice (n = 20); age-related OA was analyzed in 24-month-old mice (n = 13). Cartilage degeneration was assessed histologically using the OARSI scoring system, and synovitis was evaluated by measuring the synovial lining cell layer and the cells density in the synovial stroma. Bone changes were measured by µCT analysis. Serum levels of Postn were determined by ELISA. Expression of Postn and collagenase-3 (MMP-13) was measured by immunostaining. RNA-seq was performed on chondrocytes isolated from 21-day old Postn-/- (n = 3) and wt mice (n = 3) to discover genes and pathways altered by Postn knockout. RESULTS: Postn-/- mice exhibited significantly reduced cartilage degeneration and OARSI score relative to wt mice in post-traumatic OA after 8 weeks (maximum: 2.37 ± 0.74 vs. 4.00 ± 1.20, P = 0.011; summed: 9.31 ± 2.52 vs. 21.44 ± 6.01, P = 0.0002) and spontaneous OA (maximum: 1.93 ± 0.45 vs. 3.58 ± 1.16, P = 0.014; summed: 6.14 ± 1.57 vs. 11.50 ± 3.02, P = 0.003). Synovitis was significantly lower in Postn-/- mice than wt only in the DMM model (1.88 ± 1.01 vs. 3.17 ± 0.63; P = 0.039). Postn-/- mice also showed lower trabecular bone parameters such as BV/TV, vBMD, Tb.Th, and Tb.N and high Tb. Sp in both models. Postn-/- mice had negligible levels of serum Postn compared with wt. Immunofluorescent studies of cartilage indicated that Postn-/- mice expressed lower MMP-13 levels than wt mice. RNA-seq revealed that cell-cell-adhesion and cell-differentiation processes were enriched in Postn-/- mice, while those related to cell-cycle and DNA-repair were enriched in wt mice. CONCLUSIONS: Postn deficiency protects against DMM-induced post-traumatic and age-related spontaneous OA. RNA-seq findings warrant further investigations to better understand the mechanistic role of Postn and its potential as a therapeutic target in OA.

A Microarray Study of Articular Cartilage in Relation to Obesity and Severity of Knee Osteoarthritis.

OBJECTIVE: To query the transcript-level changes in the medial and lateral tibial plateau cartilage in tandem with obesity in patients with end-stage osteoarthritis (OA). DESIGN: Cartilage was obtained from 23 patients (20 obese [body mass index > 30 kg/m2], 3 overweight [body mass index < 30 kg/m2]) at the time of total knee replacement. Cartilage integrity was assessed using Outerbridge scale, while radiographic changes were scored on preoperative X-rays using Kellgren-Lawrence (K-L) classification. RNA was probed for differentially expressed transcripts between medial and lateral compartments using Affymetrix Gene 2.0 ST Array and validated via real-time polymerase chain reaction. Gene ontology and pathway analyses were also queried. RESULTS: Scoring of cartilage integrity by the Outerbridge scale indicated that the medial and lateral compartments were similar, while scoring by the K-L classification indicated that the medial compartment was more severely damaged than the lateral compartment. We observed a distinct transcript profile with >50% of transcripts unique between medial and lateral compartments. MMP13 and COL2A1 were more highly expressed in medial versus lateral compartment. Polymerase chain reaction confirmed expression of 4 differentially expressed transcripts. Numerous transcripts, biological processes, and pathways were significantly different between overweight and obese patients with a differential response of obesity on medial and lateral compartments. CONCLUSIONS: Our findings support molecular differences between medial and lateral compartments reflective of the greater severity of OA in the medial compartment. The K-L system better reflected the molecular results than did the Outerbridge. Moreover, the molecular effect of obesity was different between the medial and lateral compartments of the same knee plausibly reflecting the molecular effects of differential biomechanical loading.

Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Periostin (POSTN), a secretory matricellular matrix protein, plays a multitude of biologic functions. Various splice variants of POSTN have been described; however, their expression pattern and functional implications are not completely understood. This study was undertaken to decipher the differential expression pattern of POSTN and its splice variants in various tissues and cell types. We show that POSTN was more highly expressed in anterior cruciate ligament (ACL) remnants compared with articular cartilage at the cellular and tissue level. Isoforms 1 and 8 were highly expressed only in articular chondrocytes, suggesting their splice-specific regulation in chondrocytes. To discern the role of total POSTN and full-length human POSTN isoform 1 (hPOSTN-001), we stably transfected human chondrosarcoma 1 (hCh-1) cell line with hPOSTN-001 using a pcDNA3.1-hPOSTN-001 construct. RNA-sequencing analysis of hCh-1 cells identified differentially expressed genes with a known role in chondrocyte function and osteoarthritis. Similar expression of a subset of candidate genes was revealed in ACL progenitor cells and chondrocytes as well as in ACL progenitor cells in which POSTN activity was altered by overexpression and by small interfering RNA gene knockdown. Cells expressing total POSTN, not isoform 1, exhibited increased cell adhesion potential. These findings suggest an important role for POSTN in the knee.-Cai, L., Brophy, R. H., Tycksen, E. D., Duan, X., Nunley, R. M., Rai, M. F. Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Presence of meniscus tear alters gene expression profile of anterior cruciate ligament tears.

Anterior cruciate ligament (ACL) tears occur in isolation or in tandem with other intra-articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of the injured ACL is unknown. Here, we tested the hypothesis that the biological response of the ACL to injury varies based on the presence or absence of concomitant meniscus tear. We performed RNA-seq on 28 ACL tears remnants (12 isolated, 16 combined). In total, 16,654 transcripts were differentially expressed between isolated and combined injury groups at false discovery rate of 0.05. Due to the large number of differentially expressed transcripts, we undertook an Ensembl approach to discover features that acted as hub genes that did not necessarily have large fold changes or high statistical significance, but instead had high biological significance. Our data revealed a negatively correlated module containing 5,960 transcripts (down-regulated in combined injury) and a positively correlated module containing 2,260 transcripts (up-regulated in combined injury). TNS1, MEF2D, NOTCH3, SOGA1, and MLXIP were highly-connected hub genes in the negatively correlated module and SCN2A, CSMD3, LRC44, USH2A, and LRP1B were critical hub genes in the positively correlated module. Transcripts in the negatively correlated module were associated with biological adhesion, actin-filament organization, cell junction assembly, and cell matrix adhesion. The positively correlated module transcripts were enriched for neuron migration and exocytosis regulation. These findings indicate genes and pathways reflective of healing deficiency and gain of neurogenic signaling in combined ACL and meniscus tears, suggesting their diminished repair potential. The biological response of ACL to injury could have implications for healing potential of the ligament and the long term health of the knee. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2612-2621, 2018.

Advantages of RNA-seq compared to RNA microarrays for transcriptome profiling of anterior cruciate ligament tears.

Microarrays and RNA-seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA-seq and microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA-seq and microarrays and to assess potential benefits of RNA-seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time-from-injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA-seq (N = 8). The correlation of biological replicates in RNA-seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarrays values were moderate. The cross-platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA-Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA-seq and microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484-497, 2018.

Changes in Transcriptome-Wide Gene Expression of Anterior Cruciate Ligament Tears Based on Time From Injury.

BACKGROUND: Anterior cruciate ligament (ACL) tears are a common injury. The healing potential of the injured ACL is poorly understood and is considered limited. Therefore, most ACL tears that are treated surgically undergo reconstruction rather than repair. However, there has been renewed interest recently in repairing ACL tears despite unanswered questions regarding the healing capacity of the ACL. HYPOTHESIS: Gene expression in the injured ACL varies with time from injury. STUDY DESIGN: Descriptive laboratory study. METHODS: Transcriptome-wide expression profiles of 24 human ACL remnants recovered at the time of surgical reconstruction were analyzed using the Agilent human 8x60K microarray platform. Gene ontology was performed on differentially expressed transcripts based on time from injury (acute, <3 months; intermediate, 3-12 months; chronic, >12 months). A subset of transcripts with large fold changes in expression between any 2 categories was validated via microfluidic digital polymerase chain reaction. RESULTS: Numerous transcripts representing important biological processes were differentially expressed by time from injury. The most significant changes were noted between the acute and chronic groups. Expression of several extracellular matrix genes- namely, POSTN, COL5A1, COL1A1, and COL12A1-was lower in the chronic tears compared with acute and intermediate tears. In acute tears, processes representing angiogenesis and stem cell differentiation were affected. In intermediate tears, processes representing stem cell proliferation concomitant with cellular component organization/cellular localization were altered. In ACL tears more than 12 months out from injury, processes denoting myosin filament organization, cellular component organization/cell localization, and extracellular matrix organization were affected. CONCLUSION: These findings are consistent with initial repair activity in the injured ACL, which declines with time from injury. Individual genes identified in this study, such as periostin, deserve further investigation into their role in tissue repair. CLINICAL RELEVANCE: The decreased healing capacity of ACL tears over time is relevant to the development of effective techniques for repairing ACL tears and may have some significance for ACL reconstruction techniques as well. The potential for healing appears to be greatest in acute ACL tears, suggesting this window should be the focus of research for ACL repair.