Project description:Coagulation protease factor VIIa (FVIIa) is shown to induce anti-inflammatory and barrier protective effects via endothelial cell protein C receptor (EPCR)-dependent, protease-activated receptor-1 (PAR1)-mediated cell signaling. FVII-EPCR-PAR1 signaling also induces the release of extracellular vesicles from endothelial cells. To obtain clues on whether microRNA (miR) carried out by FVIIa-released EEVs contribute to anti-inflammatory and barrier protective effects, we analyzed miR expression in control- and FVIIa-released EEVs by deep sequencing. These data revealed that several anti-inflammatory miR expression was higher (more than 2-fold) in FVIIa-released EEVs compared to control EEVs, the most predominant being miR10a-5p. The differential expression of miR10a-5p and several other abundant miRs were validated by qRT-PCR. Subsequent in vitro and in vivo experiments showed that miR10a in FVIIa-released EEVs contribute to anti-inflammatory and barrier protective effects.
Project description:Coagulation protease signaling within the tumor-microenvironment (TME) causes cancer immune evasion and impairs immune checkpoint-inhibitor therapy. Tumor-associated macrophages produce factor X and drive immune-evasive protease activated receptor-2 (PAR2) signaling. Myeloid FXa-PAR2 signaling promotes the enrichment of resident-like macrophages displaying an immunosuppressive repair phenotype within the TME of mice with genetically induced spontaneous breast cancer development. Conversely, loss of FXa-PAR2 signaling favors the expansion of monocyte-derived macrophage subsets relevant for DNA-sensing, autophagy and cGAS-STING-IFNβ mediated stimulation of anti-tumor immunity. Thereby FXa suppresses the function of CD103+ dendritic cells relevant for priming, reactivation, and expansion of CD8+ T-cells, including antigen-experienced progenitor exhausted T-cells. Our findings thus emphasize the translational potential of FXa inhibition to synergize with immunotherapy.ompacted abstract
Project description:Various culture media that can rapidly expand bone marrow stromal cells (BMSCs) are currently available. However, the effects of those culture media on the contents of extracellular vesicles released by bone marrow stromal cells have not been fully understood. Using BMSCs from 6 healthy donors were cultured in two different culture media and characterized the small RNA profiles in extracellular vesicles.
Project description:Determine if transcripts are released by protease treatment During total RNA isolation cell free lysates from stationary-phase cultures or exponential cultures were treated with one of three proteases or buffer alone. All experimental samples are over a common reference. There are two replicates for each sample.
Project description:CD4+ T cells release extracellular vesicles during their differentiation process. It is known that miRNAs encapsulated within these extracellular vesicles (EVs) are involved in the regulation of cellular function and the control of disease. It has become increasingly clear that secretory PLA2 (sPLA2), an extracellular metabolic enzyme, metabolizes the membrane lipids of EVs and regulates their function. Therefore, we focused on PLA2G12A, a member of the sPLA2 family, and investigated whether its deficiency alters the miRNA profile. We isolated naive T cells from mouse spleens, differentiated them into Th17 cells using a Th17 differentiation culture system, and analyzed the EVs released during this process. Although no significant overall differences were observed between wild-type and gene-deficient, differences in expression levels were confirmed for specific miRNAs. These miRNAs were factors regulating Th17 cell differentiation. These results suggest that PLA2G12A alters the composition of EVs.
Project description:We have isolated and characterized the extracellular vesicles released by Naegleria fowleri (ATCC 30808) and determined their effect on MDCK cells and erythrocytes.
Project description:In order to identify small RNAs that are enriched in T-cell extracellular vesicles (EVs) upon T cell activation in a nSMase-dependent manner, we used a two-step ultracentrifugation procedure to isolate EVs released by T cells followed by small RNA profiling of EVs and T cells under resting, stimulated conditions and stimulated conditions in the presence of the vehicle control, DMSO, or nSMase inhibitor, GM4869.
Project description:MASLD and its more severe version, MASH, are attributable to metabolic dysfunction and are associated with cardiometabolic disease, atherothrombosis, and obesity. The exact pathophysiological mechanisms linking MASLD and CVD have yet to be elucidated but share common metabolic abnormalities that could interact synergistically to increase the risk of both heart disease and liver disease. Protease-activated receptor 2 (PAR2), a receptor activated by prothrombotic coagulation proteases that are dysregulated in inflammatory and fibrotic diseases, is emerging as a new drug target for MASH. Using patient samples, we discovered that expression of liver PAR2 is increased in the livers of obese patients with MASLD and MASH.To address the direct contribution of hepatocyte PAR2 to progression of MASLD and obesity, we fed hepatocyte-specific PAR2 knockout mice(PAR2dHep), whole-body PAR2-deficient mice(PAR2 KO), and control floxed WT mice(PAR2fl/fl) high-fat diet and conducted metabolic, biochemical and RNA-seq analyses. Hepatocyte-specific deletion of PAR2 caused a reduction in body weight and significantly lowered triglycerides and cholesterol levels in plasma and livers of mice fed high-fat diet. Analysis of bulk liver RNA seq data showed that global or hepatocyte-specific deletion of PAR2 resulted in significant improvements in fatty acid metabolic and reverse cholesterol transport pathways in liver.These data provide support that targeting hepatic PAR2 may potentially provide broad liver and cardiometabolic benefits to patients with MASLD and MASH.