Project description:Current knowledge of mechanically-induced regulation of gene expression in fibroblast is mostly based on experiments using prolonged cyclical stretching of either cultured cells or load-bearing connective tissues such as tendons and ligaments. In this study, we have examined the effect of static tissue stretch on fibroblasts within loose connective tissue using in vivo and ex vivo models. In an in vivo trunk side bending model, one side of the trunk of the mouse was stretched while the other side was shortened under anesthesia for 90 minutes. In the ex vivo method, whole subcutaneous tissue explants (including skin, subcutaneous muscle and subcutaneous tissue) from both sides of the trunk were incubated ex vivo either stretched or not stretched (shortened) for 24 hours. Tissue stretch or shortening was followed by immediate dissection of the loose connective tissue layer on both sides of the trunk, RNA extraction and gene expression analysis using Affymetrix mouse gene arrays. In both in vivo and ex vivo experiments, a large cluster of genes related to skeletal muscle function and carbohydrate metabolism was up-regulated in the stretched, compared with the shortened, connective tissue samples. However, there were few differentially expressed matrix-related genes, and in particular no change in collagen genes. Immunohistochemical staining for myosin after tissue stretch for 24 hours ex vivo showed increased myosin immunoreactivity in fibroblasts within the stretched compared with the shortened tissue samples. These results suggest that dynamic modulation of muscle-related gene expression is a normal physiological response of loose connective tissue fibroblasts to changes in tissue length.. Keywords: response to mechanical stretch Within animal stretched vs shortened (randomized): 7 animals in vivo and 4 ex vivo.
Project description:Current knowledge of mechanically-induced regulation of gene expression in fibroblast is mostly based on experiments using prolonged cyclical stretching of either cultured cells or load-bearing connective tissues such as tendons and ligaments. In this study, we have examined the effect of static tissue stretch on fibroblasts within loose connective tissue using in vivo and ex vivo models. In an in vivo trunk side bending model, one side of the trunk of the mouse was stretched while the other side was shortened under anesthesia for 90 minutes. In the ex vivo method, whole subcutaneous tissue explants (including skin, subcutaneous muscle and subcutaneous tissue) from both sides of the trunk were incubated ex vivo either stretched or not stretched (shortened) for 24 hours. Tissue stretch or shortening was followed by immediate dissection of the loose connective tissue layer on both sides of the trunk, RNA extraction and gene expression analysis using Affymetrix mouse gene arrays. In both in vivo and ex vivo experiments, a large cluster of genes related to skeletal muscle function and carbohydrate metabolism was up-regulated in the stretched, compared with the shortened, connective tissue samples. However, there were few differentially expressed matrix-related genes, and in particular no change in collagen genes. Immunohistochemical staining for myosin after tissue stretch for 24 hours ex vivo showed increased myosin immunoreactivity in fibroblasts within the stretched compared with the shortened tissue samples. These results suggest that dynamic modulation of muscle-related gene expression is a normal physiological response of loose connective tissue fibroblasts to changes in tissue length.. Keywords: response to mechanical stretch
Project description:Failure of ligamentous support of the genital tract to resist intra-abdominal pressure is a plausible underlying mechanism for the development of pelvic organ prolapse, but the nature of molecular response of pelvic tissue support remains unknown. We hypothesized that the expression of genes coding for proteins involved in maintaining the cellular and extracellular integrity would be altered in cases of pelvic organ prolapse. Therefore, cDNA microarrays were used to examine the difference in transcriptional profile in RNA of primary culture fibroblasts subjected to mechanical stretch and those that remained static. Keywords: Changes in transcription profile in pelvic organ fibroblasts in response to stretch
Project description:Although connective tissues play critical roles in development, our understanding of connective tissue fibroblast developmental programs lag behind that of other components of the musculoskeletal system mainly because fibroblasts are highly heterogeneous and poorly characterized. Combining single-cell RNA-sequencing-based strategies, including trajectory inference, and in situ hybridization analyses, we address the diversity of connective tissue fibroblasts and their developmental trajectories during chicken limb foetal development. We show that fibroblasts switch from a positional information to a lineage diversification program during their development. Muscle connective tissue and tendon contain several fibroblast populations that emerge asynchronously. In fine, these populations map to distinct layers, prefiguring the adult fibroblast layers. Populations that are close in transcriptional identity are found in neighboring layers. Altogether, we propose that the limb connective tissue is organised in a continuum of promiscuous fibroblasts identities, allowing for the robust and efficient connection of highly different tissues such as muscle, bone and skin.
Project description:Purpose : Positional information driving limb muscle patterning is considered to be contained in lateral plate mesoderm-derived tissues, such as tendon or muscle connective tissue and not in myogenic cells themselves. The current consensus is that myogenic cells originate from somites, while connective tissue fibroblasts originate from the lateral plate mesoderm. We challenged this model by cell and genetic lineage tracing experiments and identified that a subpopulation of limb myogenic cells did not originate from somite or Pax3 lineage, but rather originated from the lateral plate mesoderm and were derived from Osr1 and Scx lineages. Results: Analysis of single-cell RNA-sequencing data obtained from limb cells at successive developmental stages identified a subpopulation of cells displaying a dual muscle and connective tissue signature, in addition to independent muscle and connective tissue populations. Active BMP signalling was detected in this junctional cell sub-population and at the tendon/muscle interface in developing limbs. BMP gain- and loss-of-function experiments performed in vivo and in vitro showed that this signalling pathway regulated a fibroblast-to-myoblast conversion. Conclusions: We propose that localised BMP signalling converts a subset of lateral plate mesoderm-derived fibroblasts to a myogenic fate and establishes a boundary of fibroblast-derived myonuclei at the tendon/muscle interface to control the muscle pattern during limb development and myotendinous formation.
Project description:AVICs were exposed to cyclic stretch to examine the role of mechanical stimuli on gene expression AVICs cultured on collagen 1 coated Bioflex were exposed to 14% stretch at 1 hz or static conditions using a Flexcell-5000 14% stretch was the experimental condition while the static condition was the control
Project description:We explored the hypothesis that Serotonin (5HT) receptor signaling, that can be enhanced with 5HT transporter blockade with Fluoxetine (Fluox), in the aortic valve may vary based upon the biomechanical activity of the aortic valve leaflet. We used Affymetrix microarrays to study gene expression profiling of Porcine Aortic Valves (PAV) incubated under organ culture conditions for 24 hours in either a static state or with 10% cyclic stretch, simulating physiologic leaflet motion. PAV in the bioreactor with or without stretch were exposed to 5HT along or the combination 5HT plus Fluox. Fresh porcine aortic valves were obtained from a local abattoir. The three leaflets were excised from each valve and a rectangular section of tissue 15x10 mm was isolated from the central region of each valve cusp. These samples were randomized and assigned to one of four groups. The experimental groups were: 1) Static conditions with no agents added; 2) Cyclic stretch conditions with no agents added; 3) Static conditions with 5HT plus Fluox added; and 4) Cyclic stretch conditions with 5HT plus Fluox added.
Project description:miRNA-Sequencing was performed on human aortic valve interestitial cells (AVICs) exposed to 14% stretch at 1 hz or static conditions for 24h. Six static control and six samples exposed to cyclic stretch 14% for 24h
Project description:Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.
Project description:Vascular smooth muscle cells (VSMCs) respond to biomechanical stretch with specific changes in gene expression which govern the phenotype of these cells. The mechanotransducer zyxin is a potential candidate for regulating the expression of such genes. Using microarrays, we compared stretch-induced gene expression in wild type and zyxin-null VSMCs to define such changes in detail. Wild type (WT) and zyxin-null VSMCs were stretched at 10% cyclic elongation for 6 hours and the changes in gene expression were compared under static and stretched conditions. Up to 3 biological replicates were used for each of the 4 sample types.