Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFß/SMAD2 axis.

BACKGROUND: Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations. METHODS: EVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis. RESULTS: We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFß/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue. CONCLUSIONS: In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration.

Driver Mutations of Pancreatic Cancer Affect Ca2+ Signaling and ATP Production.

Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations-KrasG12D and Trp53R172H-present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since Ca2+ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular Ca2+ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited Ca2+ signals of different frequencies in KPC cells compared to control cells. Finally, Ca2+ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent.