Tissue Factor Pathway Inhibitor-2 is Induced by Fluid Shear Stress in Vascular Smooth Muscle Cells and Affects Cell Proliferation and Survival
Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival.
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View SamplesNeuroanatomical methods enable high-resolution mapping of neural circuitry, but do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of a novel approach for molecularly profiling projective neurons. We show that ribosomes can be labeled with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from cells expressing GFP. We generated a transgenic mouse encoding a nanobody-ribosomal protein fusion (Syn-NBL10) and used a retrograde virus (CAV) encoding GFP to immunoprecipitate ribosomes from projection neurons. This enabled us to profile neurons projecting to the nucleus accumbens. The current method provides a new means for profiling neurons based on their projections. Overall design: Translating mRNAs immunoprecipitated from neurons projecting to the nucleus accumbens. Each Input and IP sample corrspond to a pooled group of 6 mice.
Molecular profiling of neurons based on connectivity.
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View SamplesA biobank collection of carotid plaque samples taken from patients undergoing endarterectomy operations.
Prediction of ischemic events on the basis of transcriptomic and genomic profiling in patients undergoing carotid endarterectomy.
Specimen part, Disease, Subject
View SamplesWe report that phosphorylated ribosomes can be immunoprecipitated from mouse brain homogenates, resulting in enrichment of transcripts expressed in activated neurons. Overall design: Mice were either injected with a concentrated salt solution or vehicle, hypothalami dissected, and phosphorylated ribosomes immunoprecipitated. RNA was sequenced from the input and IP for each condition (4 samples total).
Molecular profiling of activated neurons by phosphorylated ribosome capture.
Specimen part, Cell line, Treatment, Subject
View SamplesTranslational profiling methodologies enable the systematic characterization of cell types in complex tissues such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy to profile CNS cell types in a spatiotemporally-restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by TRAP. We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting AAV injections enabled the elucidation of regional differences in gene expression within this cell type. Taken together, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre. Overall design: Polysome-bound mRNAs from TRAP IPs were compared to whole tissue mRNAs. Data was collected from MCH neurons in hypothalamus using vTRAP, cortical layer 6 Ntsr1 neurons using vTRAP, and cortical layer 6 Ntsr1 neurons using bacTRAP. We include vTRAP data from three AAV serotypes for the cortical Ntsr1 cells. We collected three replicates for IP and inputs for vTRAP experiments, while bacTRAP data was collected in duplicate.
Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP.
Specimen part, Subject
View SamplesExosomes are vesicles of endocytic origin released by many types of cells into the extracellular environment. In an attempt to further examine the exosome-mediated cellular communication, we show that exosomes from a mouse mast cell line (MC/9), exosomes from primary bone marrow derived mast cells, and exosomes from a human mast cell line (HMC-1) contain RNA but not DNA.
Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
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View SamplesBone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li+). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li+, while PLGA without Li+ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li+ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li+, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li+ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li+. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li+ compared with Ctrl. The presence of -catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors knowledge the first report evaluating the effect of a local release of Li+ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li+ in PLGA implants, Li+ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway.
Gene expression profiling of peri-implant healing of PLGA-Li+ implants suggests an activated Wnt signaling pathway in vivo.
Sex, Specimen part, Treatment, Time
View SamplesBackground: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes.
Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells.
Cell line
View SamplesWe isolated CD4+ T cells from draining lymph nodes 7 days post EAE from
Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis .
Sex, Specimen part
View SamplesMacrophages play a critical role in the pathogenesis of many diseases, including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Monocytes recruited into tissues from peripheral blood differentiate into macrophages. There is limited data concerning the global changes in the expression of genes during monocyte to macrophage, and how the patterns of change identify the mechanism contributing to differentiation or macrophage function. Employing the microarray technology, we examined the transcriptional profile of in vitro adherence-induced differentiation of primary human monocytes into macrophages. We found the significant up regulation of genes contributing to the functions of macrophage, including signature patterns defining the induction of genes contributing to immunity and defense; lipid, fatty acid and steroid metabolism; cell adhesion and; carbohydrate metabolism; amino acid metabolism and endocytosis. In contrast, a variety of transcription factors were down regulated during monocyte to macrophage differentiation, suggesting that transcriptional repression may be important for the transition from monocytes to macrophages. However, a limited number of transcription factors were up regulated, among these was C/EBPA, which may contribute to differentiation by regulating down stream genes, which a characteristic of differentiated macrophages. These observations suggest that examination of the transcriptional profile in monocytes and macrophages in patients may identify relevant therapeutic targets in diseases such as rheumatoid arthritis and atherosclerosis.
Transcriptional diversity during monocyte to macrophage differentiation.
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