RhoA Determines Lineage Fate of MSCs by Modulating CTGF-VEGF Complex in Extracellular Matrix
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE73557 | GEO | 2016/03/01
SECONDARY ACCESSION(S): PRJNA297328
REPOSITORIES: GEO
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Project description:Mesenchymal stem cells (MSCs) participate in the repair/remodeling of many tissues, where MSCs commit to different lineages dependent on the cues in the local microenvironment. Here we show that TGFβ-activated RhoA/ROCK functions as a molecular switch regarding the fate of MSCs in arterial repair/remodeling after injury. MSCs differentiate into myofibroblasts when RhoA/ROCK is turned on, endothelial cells when turned off. The former is pathophysiologic resulting in intimal hyperplasia, whereas the latter is physiological leading to endothelial repair. Further analysis revealed that MSC RhoA activation promotes formation of an extracellular matrix (ECM) complex consisting of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF). Inactivation of RhoA/ROCK in MSCs induces matrix metalloproteinase-3-mediated CTGF cleavage, resulting in VEGF release and MSC endothelial differentiation. Our findings uncover a novel mechanism by which cell-ECM interactions determine stem cell lineage specificity and offer additional molecular targets to manipulate MSC-involved tissue repair/regeneration. Mouse bone marrow MSCs were purchased from Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine. All MSCs were cultured in αMEM supplied with 10% FCS. The transfection of DNA plasmids were performed with Lipofectamine® LTX with Plus Reagent (Life Technologies). Myc-L63RhoA or empty vector (control) was transfected into mouse bone marrow MSCs. Three days later, the cells were harvested and total RNA was isolated using RNeasy Mini kit (Qiagen). A total of four independent experiments were performed. RNA samples were assessed for quality and integrity using Synergy HT (Biotek, Winooski, VT). Microarray expression profiles were generated using the Illumina MouseRef-8 v2.0 Expression BeadChip (Illumina, San Diego, CA). Biotin-labeled cRNA was synthesized by the total prep RNA amplification kit from Ambion (Austin, TX). cRNA was quantified and normalized to 75 ng/µl, and then 1µg was hybridized to Beadchips. The hybridized chip was scanned using the Illumina iScan system and background corrected signal intensities were extracted using the GenomeStudio software (Illumina). The lumi R package was used to transform the data using a Variance Stabilizing Transformation (VST) and normalized using quantile normalization. Differential expression analysis was performed using the R/Mannova package. P-values were calculated by performing 1000 permutations, then corrected for multiple comparisons by false-discovery rate (FDR) transformation, using a 20% FDR cutoff.
2016-03-01 | E-GEOD-73557 | biostudies-arrayexpress
Project description:The small GTPase RhoA regulates a variety of cellular processes, including cell motility, proliferation, survival and permeability. In addition, there are reports suggesting that the RhoA-ROCK axis plays a role in VEGF-mediated angiogenesis, whereas other work has shown opposite effects. To elucidate this conflicting data, we examined HUVEC and HCAEC after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a variety of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. We observed that expression of active as well as wild-type RhoA but not expression of dominant-negative RhoA significantly increased endothelial cell death as well as inhibited endothelial cell proliferation, migration, tube formation and angiogenic sprouting of endothelial cells in vitro and reduced HUVEC-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized inhibitory RhoA effects and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 had no significant effect on RhoA-related effects, but again increased angiogenic sprouting and migration of control-treated cells. In line with these data, VEGF did not activate RhoA in HUVEC as measured by a FRET-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology (GO) enrichment analyses revealed that constitutively active RhoA induces a differentially expressed gene pattern that is enriched for GO biological process terms such as mitotic nuclear division, cell proliferation, cell motility and cell adhesion and includes a significant decrease in VEGFR-2 and NOS3 expression. Thus, our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and anti-angiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.
2022-07-06 | GSE182806 | GEO
Project description:Scarcity of gender specific donor hearts highlights the importance of mesenchymal stem cells (MSCs) as a therapeutic tool for heart repair. However, inefficient incorporation, retention, and activity of MSCs in cardiac tissue remains an obstacle. Since surge in follicular estradiol (μmolar-range) triggers tissue remodeling (e.g. ovulation) and estradiol exerts beneficial actions on the cardiovascular system, we hypothesized that estradiol may promote/improve MSC-mediated cardiac repair processes. Methods: Wharton’s jelly-derived MSCs were used, to assess the effects of estradiol on their proliferation, directed-migration and engraftment in murine heart slices, ex vivo. Results: MSCs expressed estrogen receptors (ERs) α and β, and estradiol promoted MSC proliferation (measured using xCELLigence real-time cell-impedance system and DNA-quantification). Estradiol up-regulated mRNA (qRT-PCR) and protein expression (western blotting) of ERα, ERβ, EMMPRIN, and MMP-9, yet down-regulated MMP-2 expression. In MSCs estradiol, up-regulated mRNA expression of VEGF-A, VCAM-1, and angiogenin, and stimulated NO production via ER. Proteomic analysis revealed that in MSCs estradiol up-regulated 47 proteins, down-regulated 7 proteins, and increased the expression of key biochemical components/pathways involved in tissue repair. In MSCs co-cultured with murine heart-slices, estradiol significantly induced MSC migration in an ER-dependent fashion (and preferentially via ERα) and significantly increased the secretion of MMP-2, MMP-9, angiogenin and VEGF. Conclusion: Estradiol facilitates the integration/engraftment of MSCs into heart slices by promoting MSC proliferation and migration and these beneficial effects are mediated via increases in molecules/pathways involved in tissue remodeling and angiogenesis. Priming of MSCs with estradiol may enhance their ability to repair/regenerate cardiac tissue in women.
2019-06-24 | PXD011742 | Pride
Project description:Mesenchymal stem cells (MSCs) exist in many tissues and have emerged as promising candidates for therapeutic interventions. While MSCs may be used to reverse aging, they themselves also age. For example, bone marrow-derived MSCs from old individuals are known to have reduced ability to produce bone and cartilage cells, while biased towards making adipose tissues, hence aged bone marrow are referred to as "yellow marrow". Here we report, using step-wise treatment with different "cocktails" of small molecules, we turned old skin fibroblasts into much younger MSC-like cells (MSC-LCs), which reversed many aging features including shortened telomere as well as reduced proliferation potentials and differentiation bias. Moreover, MSC-LCs manifest potent effects in bone and cartilage repair in vivo. MSC-LCs also demonstrated strong immune modulatory functions both in vitro and in vivo during liver damage repair. Taken together, our study demonstrated that old skin fibroblasts could be reprogrammed to acquire youth and harbor huge therapeutic values.
2018-08-20 | GSE102983 | GEO
Project description:Efficient osteogenic differentiation of mesenchymal stem cells (MSCs) is crucial to accelerate bone formation. In this context, the use of extracellular matrix (ECM) as natural 3D-framework mimicking in vivo tissue architecture is of interest. The aim of this study was to generate a devitalized human osteogenic MSC-derived ECM and to investigate its impact on MSC osteogenic differentiation to improve MSC properties in bone regeneration. The devitalized ECM significantly enhanced MSC adhesion and proliferation. Osteogenic differentiation and mineralization of MSCs on the ECM was quicker than in standard conditions. The presence of ECM promoted in vivo bone formation by MSCs in a mouse model of ectopic-calcification. We analyzed the ECM composition by mass spectrometry, detecting 846 proteins. Of these, 473 proteins were shared with the human bone proteome we previously described, demonstrating high homology to an in vivo microenvironment. Bioinformatic analysis of the 846 proteins showed involvement in adhesion and osteogenic differentiation, confirming the ECM composition as key modulator of MSC behaviour. In addition to known ECM-components, proteomic analysis revealed novel ECM functions, which could improve culture conditions. In summary, this study provides a simplified method to obtain an in vitro MSC-derived ECM that enhances osteogenic differentiation, and could be applied as natural biomaterial to accelerate bone regeneration.
2022-02-24 | PXD006865 | Pride
Project description:The composition and stiffness of the extracellular matrix (ECM) environment that surrounds Multipotent mesenchymal stem cells (MSCs) stem cells dictates transcriptional programming, thereby affecting stem cell lineage decision-making. Cells sense force between themselves and their microenvironment controlling the capability of MSCs to differentiate into adipocytes, osteocytes and chondrocytes. Force sensing is transmitted by integrin receptors and their associated adhesion signalling complexes. To identify regulators of MSC force sensing we sought to catalogue MSC adhesion complex composition. Therefore we isolated integrin-associated adhesion complexes formed in MSCs plated on the ECM ligand fibronectin. We identifed proteins using mass spectrometry that define a MSC specific subset of adhesion complex proteins consisting of key linkages to the actin cytoskeleton together with integrin signalling and force sensing components.
2016-07-20 | MSV000079956 | MassIVE
Project description:Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels.1-5 While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals,6,7 the identity of these signals is unknown. Here, by utilizing two functionally distinct human MSC clones, we found that so-called “pericytic” MSCs secrete the pro-angiogenic neurovascular guidance molecule SLIT3,8 which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, “non-pericytic” MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs, and knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway regulates MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications. 4 samples with 2 replicates each
2013-08-18 | E-GEOD-48302 | biostudies-arrayexpress
Project description:Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton's jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared to BM-MSCs. One human BM-MSC (pooled sample of 4 independent donors), one human WJ-MSC (pooled sample of 3 independent donors), and differentiated osteocytes and adipocytes derived from BM-MSCs after 2 weeks induction.
2013-09-05 | E-GEOD-46989 | biostudies-arrayexpress
Project description:Mesenchymal stromal cells (MSCs) combined with calcineurin-NFAT (CN-NFAT) inhibitors are being tested as a treatment for graft versus host disease (GvHD). The immunosuppressive properties of MSCs seem beneficial; however, their response during fungal infection, which is an important cause of mortality in GvHD patients, is unknown. We report that MSCs phagocytose the fungal component zymosan, resulting in phosphorylation of Syk kinase, increase in cytosolic calcium levels and ultimately, in NFAT1 nuclear translocation. RNA-sequencing analysis of zymosan-treated MSCs showed that CN-NFAT inhibition affects extracellular matrix (ECM) genes, but not cytokine expression that is under the control of the NF-κB pathway. When co-culturing MSCs or decellularized MSC-ECM with human peripheral blood mononuclear cells (PBMCs), selective NFAT inhibition in MSCs decreased cytokine expression by PBMCs. These findings reveal a dual mechanism underlying the MSC response to zymosan: while NF-κB directly controls inflammatory cytokine expression, NFAT impacts immune-cell functions by regulating ECM remodelling.
2021-06-14 | GSE157981 | GEO
Project description:The composition and stiffness of the extracellular matrix (ECM) environment that surrounds Multipotent mesenchymal stem cells (MSCs) stem cells dictates transcriptional programming, thereby affecting stem cell lineage decision-making. Cells sense force between themselves and their microenvironment controlling the capability of MSCs to differentiate into adipocytes, osteocytes and chondrocytes. Force sensing is transmitted by integrin receptors and their associated adhesion signalling complexes. To identify regulators of MSC force sensing we sought to catalogue MSC adhesion complex composition. Therefore we isolated integrin-associated adhesion complexes formed in MSCs plated on the ECM ligand fibronectin. We identifed proteins using mass spectrometry that define a MSC specific subset of adhesion complex proteins consisting of key linkages to the actin cytoskeleton together with integrin signalling and force sensing components.
2015-07-20 | PXD001809 | Pride