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RNAseq of Extracellular vesicle RNAs and cellular RNAs from HPASMCs with or without TGF-b1 or BMP4 treatment

ABSTRACT: RNAseq of Extracellular vesicle RNAs and cellular RNAs from HPASMCs with or without TGF-b1 or BMP4 treatment

PROVIDER: PRJNA545533 | ENA |

REPOSITORIES: ENA

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RNAseq of Extracellular vesicle RNAs and cellular RNAs from HPASMCs with or without TGF-b1 or BMP4 treatment

Project description:Excessive TGF-β signalling has been shown to underlie pulmonary hypertension (PAH). Human pulmonary artery smooth muscle cells (HPASMCs) can release extracellular vesicles (EVs) but their content and significance have not yet been studied. Here, we analysed the content and biological relevance of HPASMC-derived EVs. We used low-input RNA-Seq to analyse the RNAs of EVs released by HPASMC under basal conditions. This data was compared to cellular RNAs. The same experiments (RNA-seq of EV and cellular RNAs) were carried out on HPASMC treated with TGF-b1 and BMP4, recapitulating pathological conditions.

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Project description:Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by clonal expansion of myeloid cells, notably megakaryocytes (MKs), and aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-b1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-b1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-b1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-b1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found increased expression of GARP in BM and spleen of mice undergoing PMF and tested the therapeutic potential of a monoclonal antibody that blocks TGF-b1 activation by GARP-expressing cells. GARP:TGF-b1 blockade reduced not only fibrosis, but also clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-b1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-g signals in the spleen, were lost upon CD8 T cell depletion. Our results suggest that selective blockade of TGF-b1 activation by GARP-expressing Tregs increase a CD8 T cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

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Gene expression after deletion of Bmp4 in the mouse optic vesicle

Project description:Bmp4 was conditionally deleted from the mouse optic vesicle with Rx-Cre. The heads of wild type and knockout E10.5 mouse embryos were laser micodissected to isolate the lens ectoderm and prospective retina from three embryos of each genotype. Total RNA was purified and reverse transcribed and amplified using a NuGEN Ovation Pico WTA system V2 kit. cDNA was biotinylated and hybridized to Illumina Mouse6 V2 bead arrays. Three wild type and three knockout embryos were used. RNA obtained from the lens ectoderm or prospective retina (distal optic vesicle) was pooled, amplified and used for microarray analysis. Statistical analysis was performed using the values from the 25-50 beads on each array.

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