Transcriptomic Analysis of Cellular Pathways during Healing of Flexor Tendons in Plasminogen Activator Inhibitor Type 1, Member 1 (PAI-1/Serpine1) Null Mice
Ontology highlight
ABSTRACT: Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Adhesions have been associated with PAI-1, a master suppressor of protease activity including matrix metalloproteinases (MMP). In the present study, we used next generation RNA sequencing (RNA-seq) to assess genome-wide differences in mRNA expression due to PAI-1 deficiency after zone II flexor tendon injury. Ingenuity pathway analysis was used to characterize molecular pathways and biological drivers associated with differentially expressed genes. Analysis of hundreds of overlapping and differentially-expressed genes in PAI-1 knockout and C57Bl/6J mice during tendon healing revealed common and distinct biological processes associated with the regulation of matrix organization, cell cycle, and immune response. Most importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1-associated biological processes as a unique transcriptional signature of the healing tendon in the PAI-1 KO mice. The differences in transcriptomics between the two mouse strains are transcriptionally related to complex cross-talk between PI3K/Akt/mTOR, PKC, and MAPK signaling cascades that drive differences in transcriptional regulation of cell proliferation, survival and senescence, and chronic inflammation as potential drivers of fibrotic healing and adhesions in the C57Bl/6J injured tendons. These transcriptional observations should guide future studies to develop an improved understanding of the biological limitations of tendon healing as the basis for rational design of targeted therapeutics for scar-free regenerative healing of tendon.
Project description:Purpose: The goal of this study is to compare transcriptional profiles of flexor tendon healing in wild-type (WT, C57Bl/6J) to superhealer (MRL/MpJ) miceto gain insights in the biological drivers of the tendon injury response between the C57 and MRL mice. Methods: RNA was isolated from patially lacerated or uninjured flexor tendon 7 days post-injury. Results: Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Conclusions: There is altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.
Project description:Little is understood about the roles of tendon cells during flexor tendon healing. To better understand tendon cell functions, the Scx-Cre mouse was crossed to the DTR mouse model to facilitate scleraxis lineage cell depletion prior to acute flexor tendon injury and repair. WT (cre-) and experimental (cre+) mice underwent complete transection and repair of the flexor digitorum longus tendon. Repaired tendons were harvested at 14 and 28 days post-repair for bulk RNA-Seq analysis to examine possible mechanisms driving differential healing due to Scx lineage cell depletion.
Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data.
Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Computed
Project description:Vascular inflammation and myofibroblast activation by TGF-β1 are key, yet-to-be-fully-understood pathological processes in fibrosis. Here we report the development of a novel human Tendon-on-a-Chip (hToC) to elucidate the role of TGF-β1 in peritendinous adhesions, a debilitating fibrosis condition affecting flexor tendon, which currently lacks biological therapies. The hToC allows the crosstalk between a vascular compartment harboring endothelial cells and monocytes with a tissue hydrogel compartment containing tendon fibroblasts and macrophages. We find that the hToC replicates in vivo inflammatory and fibrotic phenotypes in preclinical and clinical samples, including myofibroblast differentiation and tissue contraction, excessive ECM deposition, and inflammatory cytokines secretion. We further show the fibrotic phenotypes are driven by the interactions between the vascular and tissue compartments, mediating the transmigration of monocytes. We demonstrate significant overlap in fibrotic transcriptional signatures in the hToC with human tenolysis samples, including the mTOR pathway, a regulatory nexus of fibrosis across various organs. Treatment with Rapamycin suppressed the fibrotic phenotype on the hToC, which validates the hToC as a preclinical alternative for investigating fibrosis and testing therapeutics.
Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Keywords: time_series_design
Project description:Impaired healing of adult tendons with fibrosis remains clinical challenges while neonatal tendons have full functional restoration. However, age-associated cellular and molecular changes in tendon cells and tendon stem/progenitor cells (TSPCs) remain unknown. Here, comparative single cell transcriptomics of early postnatal (2-week-old) and adult (20-week-old) mouse tendons revealed that adult tendons have reduced number of TSPCs, decreased gene expression in tendon and cartilage development, and a greater population of fibro-tenogenic cells. Notably, adult TSPCs and tenocytes exhibit increased expression of immune-response and oxidative-stress genes with higher EGFR but decreased IGF signaling. Adult tendon cells show increased levels of intracellular reactive oxygen species (ROS) in vivo. In contrast, antioxidant treatment of adult tendons significantly reduces intracellular ROS of TSPCs and improves tendon strength in vivo. Hence, these findings suggest that increased inflammation and ROS during tendon aging deteriorates tendon function and regeneration that can be mitigated by antioxidant treatment.
Project description:Aged tendons have disrupted homeostasis, increased injury risk, and impaired healing capacity. Understanding mechanisms of homeostatic disruption is crucial for developing therapeutics to retain tendon health through the lifespan. Here, we developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxislineage (ScxLin) cells in young mice (DTR). DTR recapitulates many aspects of tendon aging including comparable declines in cellularity, alterations in ECM structure, organization, and composition. Single cell RNA-sequencing demonstrated a conserved decline in tenocytes associated with ECM biosynthesis in aged and DTR tendons, identifying the requirement for ScxLin cells during homeostasis. However, the remaining cells in aged and DTR tendons demonstrate functional divergence. Aged tenocytes become pro-inflammatory and lose proteostasis. In contrast, DTR tenocytes demonstrate enhanced remodeling capacity. Collectively, this study defines DTR a novel model of accelerated tendon ECM aging and identifies novel biological intervention points to maintain tendon function through the lifespan
Project description:Spatial transcriptomic analysis of murine FDL tendons following acute injury and repair to evaluate spatiotemporal programming of tendon healing
Project description:MicroRNA has the potential for cross-regulation and functional integration of discrete biological processes during complex physiological events. In this study, we found that the highly expressed microRNAs in exosomes from bone marrow derived macrophage appear to control fibrotic healing response in the tendon. Notably, fibrotic microRNA-21 in mice distributed in the early stage of healing after tendon injury, having the similar expression with mammals. Therefore, we hypothesized the bone marrow derived macrophage secreted miRNAs-containing exosomes play important functions in peritendinous adhesion after tendon injury.