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

(2023) 21:412 Paw et al. BMC Medicine https://doi.org/10.1186/s12916-023-03117-w RESEARCH ARTICLE BMC Medicine Open Access Hypoxia enhances anti‑fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b‑3p/TGFβ/SMAD2 axis Milena Paw1, Agnieszka A. Kusiak1,2, Kinga Nit1, Jacek J. Litewka1,2, Marcin Piejko3, Dawid Wnuk1, Michał Sarna4, Kinga Fic5, Kinga B. Stopa2,5, Ruba Hammad6,7, Olga Barczyk‑Woznicka8, Toni Cathomen6,7, Ewa Zuba‑Surma1, Zbigniew Madeja1, Paweł E. Ferdek1 and Sylwia Bobis‑Wozowicz1*    Abstract Background Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unad‑ dressed 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 extra‑ cellular 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 con‑ centration (hypoxia): 3% O2 (EV-H3) or 5% O 2 (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 com‑ pared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFβ/SMAD pathway, primarily via miR302b-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 antifibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration. Keywords Extracellular vesicles, Induced pluripotent stem cells, Hypoxia, Low oxygen, Heart fibrosis, Therapy *Correspondence: Sylwia Bobis‑Wozowicz Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑ mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Paw et al. BMC Medicine (2023) 21:412 Page 2 of 25 Graphical Abstract Background The incidence of cardiovascular diseases has increased dramatically in the last decades and remains the predominant cause of mortality worldwide [1, 2]. Although myocardial dysfunction during heart diseases is often associated with impaired cardiomyocyte activity, cardiac fibrosis is the major cause of end-stage heart damage [3, 4]. Myocardial fibrosis is a complex process that results from abnormal healing of the heart tissue and ultimately leads to the formation of a non-functional scar, which hampers the activity of the entire organ. This involves both extracellular matrix overproduction and the activation of structural non-excitable fibroblasts to differentiate into contractile myofibroblasts, in a process called fibroblast-to-myofibroblast transition (FMT). Myofibroblasts are characterized by an increased expression of α-smooth muscle actin (α-SMA, encoded by the ACTA2 gene) and the secretion of a number of pro-fibrotic proteins, such as collagens, fibronectin, or tenascin [5–7]. Transforming growth factor β (TGFβ) is the best-known fibrogenic cytokine described in fibrotic diseases, including heart fibrosis [6, 8, 9]. By enhanced activation of multiple signaling pathways, particularly those involving SMAD2/3 proteins, TGFβ is able to effectively induce the FMT machinery and propagate the profibrotic signaling cascade [3, 6]. Currently used therapeutic strategies targeting cardiac fibrosis such as β-blockers or cell-based therapies neither prevent the progression of cardiac fibrosis nor promote the functional recovery of the heart [10]. Therefore, innovative treatment strategies are an important unmet clinical need. One of the novel classes of therapeutics which has gained considerable interest in recent years constitutes extracellular vesicles (EVs). EVs are nanometric circular structures secreted by virtually all types of cells under physiological and pathological conditions. They contain bioactive components derived from the parental cell, enclosed in a lipid bilayer that protects them against rapid degradation [11]. Based on their size and origin, EVs can be classified as exosomes, microvesicles (shedding vesicles), and apoptotic bodies. EVs are considered to be important mediators of intercellular communication and, due to their ability to transport bioactive molecules such as proteins, lipids, and various RNA molecules, they can influence the phenotype and properties of other cells [12]. In particular, mesenchymal stem/stromal cells (MSCs), cardiac progenitor cells (CPCs), cardiospheres, endothelial cells and pluripotent stem cells were shown to produce EVs with reparative capabilities, including anti-fibrotic activity [10, 13]. Owing to their unmatched functionality, biocompatibility, and efficiency in delivering components to target cells, EVs are regarded as new generation therapeutics in the treatment of a variety of human diseases [14]. We have previously shown that EVs d (...truncated)