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Transcriptome profiling of digital flexor tendons after injury in a chicken model.

ABSTRACT: BACKGROUND:Modulation of tendon healing remains a challenge because of our limited understanding of the tendon repair process. Therefore, we performed the present study to provide a global perspective of the gene expression profiles of tendons after injury and identify the molecular signals driving the tendon repair process. RESULTS:The gene expression profiles of flexor digitorum profundus tendons in a chicken model were assayed on day 3, weeks 1, 2, 4, and 6 after injury using the Affymetrix microarray system. Principal component analysis (PCA) and hierarchical cluster analysis of the differentially expressed genes showed three distinct clusters corresponding to different phases of the tendon healing period. Gene ontology (GO) analysis identified regulation of cell proliferation and cell adhesion as the most enriched biological processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the cytokine-cytokine receptor interaction and extracellular matrix (ECM)-receptor interaction pathways were the most impacted. Weighted gene co-expression network analysis (WGCNA) demonstrated four distinct patterns of gene expressions during tendon healing. Cell adhesion and ECM activities were mainly associated with genes with drastic increase in expression 6 weeks after injury. The protein-protein interaction (PPI) networks were constructed to identify the key signaling pathways and hub genes involved. CONCLUSIONS:The comprehensive analysis of the biological functions and interactions of the genes differentially expressed during tendon healing provides a valuable resource to understand the molecular mechanisms underlying tendon healing and to predict regulatory targets for the genetic engineering of tendon repair. Tendon healing, Adhesion, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Weighted Gene Co-expression Network Analysis, Protein-protein Interaction.

SUBMITTER: Mao WF

PROVIDER: S-EPMC7276521 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Transcriptome profiling of digital flexor tendons after injury in a chicken model.

Mao Wei Feng WF Yu Yin Xian YX Chen Chen C Wu Ya Fang YF

Bioscience reports 20200601 6

<h4>Background</h4>Modulation of tendon healing remains a challenge because of our limited understanding of the tendon repair process. Therefore, we performed the present study to provide a global perspective of the gene expression profiles of tendons after injury and identify the molecular signals driving the tendon repair process.<h4>Results</h4>The gene expression profiles of flexor digitorum profundus tendons in a chicken model were assayed on day 3, weeks 1, 2, 4, and 6 after injury using t ...[more]

PMID: 32432656

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Project description:It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3 cm regions) for histologic (scoring and immunohistochemistry) and gene expression (real time PCR) analysis of extracellular matrix changes. The histopathology score was significantly higher in transected tendons compared to control tendons in all regions except for the most distal (P ? 0.03) with no differences between overstressed (medial) and stress-deprived (lateral) tendon halves. Proteoglycan scores were increased by transection in all but the most proximal region (P < 0.02), with increased immunostaining for aggrecan, biglycan and versican. After correcting for location within the tendon, gene expression for aggrecan, versican, biglycan, lumican, collagen types I, II and III, MMP14 and TIMP1 was increased in transected tendons compared with control tendons (P < 0.02) and decreased for ADAMTS4, MMP3 and TIMP3 (P < 0.001). Aggrecan, biglycan, fibromodulin, and collagen types I and III expression positively correlated with all histopathology scores (P < 0.001), whereas lumican, ADAMTS4 and MMP14 expression positively correlated only with collagen fiber malalignment (P < 0.001). In summary, histologic and associated gene expression changes were significant and widespread six weeks after injury to the equine SDFT, suggesting rapid and active development of tendinopathy throughout the entire length of the tendon. These extensive changes distant to the focal injury may contribute to poor functional outcomes and re-injury in clinical cases. Our data suggest that successful treatments of focal injuries will need to address pathology in the entire tendon, and that better methods to monitor the development and resolution of tendinopathy are required.

Project description:BackgroundLong-term cyclic tensile testing with equine palmar/plantar tendons have not yet been performed due to problems in fixing equine tendons securely and loading them cyclically. It is well established that the biomechanical response of tendons varies during cyclic loading over time. The aim of this study was to develop a clamping device that enables repetitive cyclic tensile testing of equine superficial digital flexor tendon for at least 60 loading cycles and for 5 min.ResultsA novel cryo-clamp was developed and built. Healthy and collagenase-treated pony SDFTs were mounted in the custom-made cryo-clamp for the proximal tendon end and a special clamping device for the short pastern bone (os coronale). Simultaneously with tensile testing, we used a biplanar high-speed fluoroscopy system (FluoKin) to track tendon movement. The FluoKin system was additionally validated in precision measurements. During the cyclic tensile tests of the SDFTs, the average maximal force measured was 325 N and 953 N for a length variation of 2 and 4 % respectively. The resulting stress averaged 16 MPa and 48 MPa respectively, while the modulus of elasticity was 828 MPa and 1212 MPa respectively. Length variation of the metacarpal region was, on average, 4.87 % higher after incubation with collagenase. The precision of the FluoKin tracking was 0.0377 mm, defined as the standard deviation of pairwise intermarker distances embedded in rigid bodies. The systems accuracy was 0.0287 mm, which is the difference between the machined and mean measured distance.ConclusionIn this study, a good performing clamping technique for equine tendons under repetitive cyclic loading conditions is described. The presented cryo-clamps were tested up to 50 min duration and up to the machine maximal capacity of 10 kN. With the possibility of repetitive loading a stabilization of the time-force-curve and changes of hysteresis and creep became obvious after a dozen cycles, which underlines the necessity of repetitive cyclical testing. Furthermore, biplanar high-speed fluoroscopy seems an appropriate and highly precise measurement tool for analysis of tendon behaviour under repetitive load in equine SDFTs.

Project description:BACKGROUND: Although many QTL for various traits have been mapped in livestock, location confidence intervals remain wide that makes difficult the identification of causative mutations. The aim of this study was to test the contribution of microarray data to QTL detection in livestock species. Three different but complementary approaches are proposed to improve characterization of a chicken QTL region for abdominal fatness (AF) previously detected on chromosome 5 (GGA5). RESULTS: Hepatic transcriptome profiles for 45 offspring of a sire known to be heterozygous for the distal GGA5 AF QTL were obtained using a 20 K chicken oligochip. mRNA levels of 660 genes were correlated with the AF trait. The first approach was to dissect the AF phenotype by identifying animal subgroups according to their 660 transcript profiles. Linkage analysis using some of these subgroups revealed another QTL in the middle of GGA5 and increased the significance of the distal GGA5 AF QTL, thereby refining its localization. The second approach targeted the genes correlated with the AF trait and regulated by the GGA5 AF QTL region. Five of the 660 genes were considered as being controlled either by the AF QTL mutation itself or by a mutation close to it; one having a function related to lipid metabolism (HMGCS1). In addition, a QTL analysis with a multiple trait model combining this 5 gene-set and AF allowed us to refine the QTL region. The third approach was to use these 5 transcriptome profiles to predict the paternal Q versus q AF QTL mutation for each recombinant offspring and then refine the localization of the QTL from 31 cM (100 genes) at a most probable location confidence interval of 7 cM (12 genes) after determining the recombination breakpoints, an interval consistent with the reductions obtained by the two other approaches. CONCLUSION: The results showed the feasibility and efficacy of the three strategies used, the first revealing a QTL undetected using the whole population, the second providing functional information about a QTL region through genes related to the trait and controlled by this region (HMGCS1), the third could drastically refine a QTL region.

Project description:BACKGROUND: Starvation triggers a complex array of adaptative metabolic responses including energy-metabolic responses, a process which must imply tissue specific alterations in gene expression and in which the liver plays a central role. The present study aimed to describe the evolution of global gene expression profiles in liver of 4-week-old male chickens during a 48 h fasting period using a chicken 20 K oligoarray. RESULTS: A large number of genes were modulated by fasting (3532 genes with a pvalue corrected by Benjamini-Hochberg < 0.01); 2062 showed an amplitude of variation higher than +/- 40% among those, 1162 presented an human ortholog, allowing to collect functional information. Notably more genes were down-regulated than up-regulated, whatever the duration of fasting (16 h or 48 h). The number of genes differentially expressed after 48 h of fasting was 3.5-fold higher than after 16 h of fasting. Four clusters of co-expressed genes were identified by a hierarchical cluster analysis. Gene Ontology, KEGG and Ingenuity databases were then used to identify the metabolic processes associated to each cluster. After 16 h of fasting, genes involved in ketogenesis, gluconeogenesis and mitochondrial or peroxisomal fatty acid beta-oxidation, were up-regulated (cluster-1) whereas genes involved in fatty acid and cholesterol synthesis were down-regulated (cluster-2). For all genes tested, the microarray data was confirmed by quantitative RT-PCR. Most genes were altered by fasting as already reported in mammals. A notable exception was the HMG-CoA synthase 1 gene, which was up-regulated following 16 and 48 h of fasting while the other genes involved in cholesterol metabolism were down-regulated as reported in mammalian studies. We further focused on genes not represented on the microarray and candidates for the regulation of the target genes belonging to cluster-1 and -2 and involved in lipid metabolism. Data are provided concerning PPARa, SREBP1, SREBP2, NR1H3 transcription factors and two desaturases (FADS1, FADS2). CONCLUSION: This study evidences numerous genes altered by starvation in chickens and suggests a global repression of cellular activity in response to this stressor. The central role of lipid and acetyl-CoA metabolisms and its regulation at transcriptional level are confirmed in chicken liver in response to short-term fasting. Interesting expression modulations were observed for NR1H3, FADS1 and FADS2 genes. Further studies are needed to precise their role in the complex regulatory network controlling lipid metabolism.

Project description:Autologous protein solution (APS) has been used anecdotally for intralesional treatment of tendon and ligament injuries, however, its use in these injuries has never been studied in vivo. Our objective was to evaluate the effect of APS on tendon healing in an equine superficial digital flexor (SDF) tendonitis model. We hypothesized intralesional injection of APS would result in superior structural and biomechanical healing. SDF tendonitis was induced in both forelimbs of eight horses using collagenase injection. One forelimb was randomly assigned to receive an intralesional injection of APS, while the other was injected with saline. Ultrasonographic examinations were performed at weeks -1, 0, 2, 4, 8, and 12 following treatment. At 12 weeks, horses were euthanized and SDF samples harvested. Histologic evaluation, biomechanical testing, gene expression analysis, total glycosaminoglycan (GAG) and total DNA quantification were performed. Collagen type III (COL3A1) expression was significantly higher (p = 0.028) in saline treated tendon than in normal tendon. Otherwise, there were no significant differences in gene expression. There were no significant differences in histologic or ultrasonographic scores between groups. Mean total DNA content was significantly higher (p = 0.024) in saline treated tendons than normal tendons, whereas total DNA content was not significantly different between APS treated tendon and normal tendon. Elastic modulus was higher in APS treated than saline treated tendon, but the difference was not significant. Reduced expression of COL3A1 in APS treated tendon may indicate superior healing. Increased total DNA content in saline treated tendon may indicate ongoing healing processes, vs. APS treated tendons which may be in the later stages of healing. Limitations include a relatively short study period and inconsistency in size and severity of induced lesions. Intralesional injection of APS resulted in some improvements in healing characteristics.

Dataset Information

Transcriptome profiling of digital flexor tendons after injury in a chicken model.

Publications

Transcriptome profiling of digital flexor tendons after injury in a chicken model.

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