In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE81491 | GEO | 2016/05/17
SECONDARY ACCESSION(S): PRJNA321850
REPOSITORIES: GEO
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Project description:We developed in vivo reprogramming of myofibroblasts (MFs) into induced hepatocytes (MF-iHeps) using adeno-associated virus serotype 6 (AAV6) vectors expressing hepatic transcription factors in MF fate tracing (Lrat-Cre;R26R-ZsGreen) mice with carbon tetrachloride (CCl4)-induced liver fibrosis. To determine whether MF-iHeps acquire full hepatocyte differentiation, we used microarrays to profile their global gene expression. We isolated MF-iHeps and primary hepatocytes (Heps) from the same mice by laser-capture microdissection (5 and 3 biological replicates, respectively) and hepatic MFs from CCl4-treated littermates isolated by fluorescence-activated cell sorting (3 biological replicates). Total RNA was extracted, transcribed, amplified and biotin labeled. Labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix).
2016-05-17 | E-GEOD-81491 | biostudies-arrayexpress
Project description:Myofibroblasts (MFs) are crucial components of the fibrotic remodeling after myocardial infarction (MI). We have previously demonstrated the centrality of AMPKα1 in post-MI remodeling. Here, we investigate the effects of MF-specific deletion of AMPKα1 on left ventricular (LV) adaptation following MI, and the underlying molecular mechanisms. Importantly, MF-restricted AMPKα1 conditional knockout (cKO) hearts exhibit exacerbated post-MI adverse LV remodeling and are characterized by exaggerated fibrotic response, compared to wild-type (WT) hearts. Myofibroblast proliferation significantly increases in cKO infarcted hearts, coincident with a significant reduction of Connexin 43 (Cx43) expression in MFs. Mechanistically, lack of AMPKα1 in MFs enhances miR-125b expression, which, in turn, downregulates Cx43. Collectively, our data demonstrate a cardinal role for MF-AMPKα1 in cardiac remodeling, as its lineage-specific inactivation accentuates fibrosis and LV dilatation following MI. The deleterious effects of MF-specific AMPKα1 deletion are mediated via Cx43, and its post-transcriptional regulation by miR-125b.
2020-03-25 | GSE147470 | GEO
Project description:Based on the identification of a transcriptomic signature, including Slit2, characterizing portal mesenchymal stem cells (PMSC) and derived myofibroblast (MF), we examined the gene expression profile of in liver tissue derived from multiple human liver disorders, including primary sclerosing cholangitis (PSC) (n=12), non-alcoholic steatohepatitis (NASH) (n=7) and other liver diseases (i.e., primary biliary cholangitis, autoimmune hepatitis, alcoholic liver disease and haemochromatosis) (n=8) and compared them to healthy controls (tumor free tissue from livers with metastasis from colorectal cancer) (n=5). We found that SLIT2 was overexpressed in the liver of patients with NASH, PSC and other chronic liver diseases. We also examined the microarray data of the human liver tissue samples for the transcriptomic signatures and found that in the different types of liver diseases the gene signature of PMSCs/PMSC-MFs was increased compared to normal liver, and correlated with the expression of ACTA2, COL1A1 and vWF.
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Project description:We performed transcriptomic studies comparing phenotypically defined PMSCs (PDGFRa/CD34/CD9high:CD200low) to HSCs (Vitamin-A fluorescent) of high purity, using bulk RNAseq. We further extended bulk RNAseq analyses of the two cell populations to myofibroblasts derived from both cell types in culture, including an early and late stage of differentiation for PMSC-MFs. Comparative analyses of gene expression indicated that 100 genes in PMSCs and 112 genes in HSCs, i) were overexpressed according to both scRNAseq and bulk RNAseq analyses, and ii) remained differentially expressed in these cells throughout their myofibroblastic differentiation. We selected the most differentially expressed of these genes, to build a multigene expression signature of PMSCs/PMSC-MFs (7 genes) and of HSCs/HSC-MFs (6 genes).
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Project description:The widespread use of electricity raises the question of whether or not 50 Hz (power line frequency in Europe) magnetic fields (MFs) affect organisms. We investigated the transcription of Escherichia coli K-12 MG1655 in response to extremely low-frequency (ELF) MFs. Fields generated by three signal types (sinusoidal continuous, sinusoidal intermittent, and power line intermittent; all at 50 Hz, 1 mT), were applied and gene expression was monitored at the transcript level using an Affymetrix whole-genome microarray. Bacterial cells were grown continuously in a chemostat (dilution rate D = 0.4 h-1) fed with glucose-limited minimal medium and exposed to 50 Hz MFs with a homogenous flux density of 1 mT. For all three types of MFs investigated, neither bacterial growth (determined using optical density) nor culturable counts were affected. Likewise, no statistically significant change (fold-change > 2, P ≤ 0.01) in the expression of 4,358 genes and 714 intergenic regions represented on the gene chip was detected after MF exposure for 2.5 h (1.4 generations) or 15 h (8.7 generations). Moreover, short-term exposure (8 min) to the sinusoidal continuous and power line intermittent signal neither affected bacterial growth nor showed evidence for reliable changes in transcription. In conclusion, our experiments did not indicate that the different tested MFs (50 Hz, 1 mT) affected the transcription of E. coli.
2012-01-28 | GSE35371 | GEO
Project description:Geomagnetic field (GMF) has been present since the beginning of plant evolution. Recently, some researchers have focused their efforts on employing magnetic fields (MF) higher than GMF to improve the seed germination, growth and harvest of agriculturally important crop plants, as MFs are inexpensive and environment friendly technique. In this study, we have employed different treatments considering MF of 7 mT (milliTesla) for different time point of the exposure including 1, 3, and 6 h as treatment, following longest exposure for 5 consecutive days, 6h per day in barely seeds. The results showed a positive impact of MF on growth characteristics for 5 days old seedlings including seed germination rate, root and shoot length and biomass weight, however, significant effects observed in long exposure. Moreover, ~5 day’s delay of flowering in pretreated plants was observed. We have used a shotgun proteomics approach to identify changes in the protein signatures of root and shoot tissues under MF effects. In total, we identified 2896 proteins. Thirty eight proteins in shoot and 15 proteins in root showed significant changes under MF effect. Proteins involved in primary metabolic pathways were increased in contrast to the proteins with metal ion binding function, proteins contain iron ion in their structure and proteins involved in electron transfer chain were decreased significantly in treated tissues. The prevalent biological processes of the up-regulated proteins were carbohydrate metabolic process, oxidation-reduction process and cell redox homeostasis, while down regulated processes include translation and protein refolding. In general, shoot response was more significant to MF effect compared with root tissue leading to the identification of 41 shoot specific proteins. This study provides a comprehensive view of proteome regulation in response to MF during early stage of growth and development in barley.
2021-09-09 | PXD022079 | Pride
Project description:Stable atherosclerotic plaques are characterized by a thick extracellular matrix (ECM)-rich fibrous cap populated by protective ACTA2+ myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMC). Herein, we show that in murine and human lesions, ~20-40% of ACTA2+ fibrous caps cells, respectively, are derived from non-SMC sources, including endothelial cells (EC) or macrophages that have undergone Endothelial-to-Mesenchymal (EndoMT) or Macrophage-to-Mesenchymal (MMT) transitions. In addition, weshow that SMC-specific knockout of the platelet-derived growth factor receptor beta (PDGFRB) in Apoe-/- mice fed a Western diet (WD) for 18 weeks results in brachiocephalic artery (BCA) lesions nearly devoid of SMC. While absence of SMCs does not affect lesion size, remodeling, or ACTA2+ fibrous cap cell content, prolonged WD feeding results in reduced indices of stability, indicating that EndoMT and MMT-derived MFs cannot compensate indefinitely for loss of SMC-derived MFs. Using RNA-seq analysis of the BCA region and in vitro models, we demonstrate that SMC to MF transitions (SMC-MFT) is induced by PDGF and TFGβ and is dependent on aerobic glycolysis, while EndoMT is induced by IL1β and TGFβ. Together, we provide the first quantitative evidence that the ACTA2+ fibrous cap originates from a tapestry of cell types, which transition to an MF state through distinct signaling pathways that are either dependent on or associated with extensive metabolic reprogramming.
2021-02-22 | GSE163519 | GEO
Project description:The widespread use of electricity raises the question of whether or not 50 Hz (power line frequency in Europe) magnetic fields (MFs) affect organisms. We investigated the transcription of Escherichia coli K-12 MG1655 in response to extremely low-frequency (ELF) MFs. Fields generated by three signal types (sinusoidal continuous, sinusoidal intermittent, and power line intermittent; all at 50 Hz, 1 mT), were applied and gene expression was monitored at the transcript level using an Affymetrix whole-genome microarray. Bacterial cells were grown continuously in a chemostat (dilution rate D = 0.4 h-1) fed with glucose-limited minimal medium and exposed to 50 Hz MFs with a homogenous flux density of 1 mT. For all three types of MFs investigated, neither bacterial growth (determined using optical density) nor culturable counts were affected. Likewise, no statistically significant change (fold-change > 2, P ≤ 0.01) in the expression of 4,358 genes and 714 intergenic regions represented on the gene chip was detected after MF exposure for 2.5 h (1.4 generations) or 15 h (8.7 generations). Moreover, short-term exposure (8 min) to the sinusoidal continuous and power line intermittent signal neither affected bacterial growth nor showed evidence for reliable changes in transcription. In conclusion, our experiments did not indicate that the different tested MFs (50 Hz, 1 mT) affected the transcription of E. coli. A total of 60 chips are included in this study. Each comparison (treated vs. sham-treated cultures; untreated negative control cultures) consists of three replicates. The three magnetic field test conditions include sinusoidal continous, sinusoidal intermittent (2 min on, 4 min off) and power line intermittent (2 min on, 4 min off). Exposure periods were 8 min, 2.5 h, or 15 h. The response control consists of E. coli cells treated for 10 min with 1 mM H2O2 or the equivalent water volume. The negative control comprises samples taken from cultures grown with the exposure chambers swiched off.
2012-01-28 | E-GEOD-35371 | biostudies-arrayexpress
Project description:Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine, but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert profibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease. Whole Mouse Genome Oligo Microarray v2 (4x44K) (Agilent Technologies) was used to characterize global gene expression profiles of iHeps compared to myofibroblasts and primary mouse hepatocytes. All microarrays were performed at the Research Core Unit Transcriptomics of the Hanover Medical School. Briefly, total RNA was used to prepare the aminoallyl-UTPmodified (aaUTP) cRNAs (Amino Allyl MessageAmp⢠II Kit; #AM1753; Life Technologies) as directed by the company. The aaUTP-cRNAs were labelled with Alexa Fluor 555 Reactive Dye (#A32756; LifeTechnologies). Prior to the reverse transcription reaction, 1μl of a 1:5000 dilution of Agilentâs One-Color spike-in Kit stock solution (#5188-5282, Agilent Technologies) was added to 100ng of total RNA of each analyzed sample. The cRNA fragmentation, hybridization, and washing steps were carried out according to Agilentâs One-Color Microarray-Based Gene Expression Analysis Protocol V5.7 except that 500ng of each labelled cRNA sample were used for hybridization. Slides were scanned on the Agilent Micro Array Scanner G2565 CA (pixel resolution 5 μm, bit depth 20). Data extraction was performed with the Feature Extraction Software V10.7.3.1. 12 samples were analyzed: Pr-mHSC: Primary myofibroblasts derived from hepatic stellate cells (HSCs), 2 replicates; In-vivo iHep: In-vivo myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates; In-vivo eHep: In-vivo endogenous hepatocytes (eHep), 3 replicates; In-vitro iHep: In-vitro myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates; PH24h: Primary hepatocytes (PH) cultured for 24 hours, 1 replicate.
2016-02-22 | E-GEOD-76843 | biostudies-arrayexpress