ABSTRACT
Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.
Subject(s)
Extracellular Vesicles , Mesenchymal Stem Cells , Myocardial Infarction , Myocytes, Cardiac , Extracellular Vesicles/metabolism , Extracellular Vesicles/transplantation , Humans , Animals , Mice , Myocardial Infarction/therapy , Myocardial Infarction/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Male , Myocardial Reperfusion Injury/therapy , Myocardial Reperfusion Injury/metabolism , Disease Models, Animal , Neovascularization, Physiologic , Cells, CulturedABSTRACT
Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells' response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs.
Subject(s)
COVID-19 , Extracellular Vesicles , Mice , Animals , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , COVID-19/metabolism , Extracellular Vesicles/metabolismABSTRACT
Cardiac hypertrophy is a condition that may contribute to the development of heart failure. In this study, we compare the gene-expression patterns of our in vitro stem-cell-based cardiac hypertrophy model with the gene expression of biopsies collected from hypertrophic human hearts. Twenty-five differentially expressed genes (DEGs) from both groups were identified and the expression of selected corresponding secreted proteins were validated using ELISA and Western blot. Several biomarkers, including CCN2, THBS1, NPPA, and NPPB, were identified, which showed significant overexpressions in the hypertrophic samples in both the cardiac biopsies and in the endothelin-1-treated cells, both at gene and protein levels. The protein-interaction network analysis revealed CCN2 as a central node among the 25 overlapping DEGs, suggesting that this gene might play an important role in the development of cardiac hypertrophy. GO-enrichment analysis of the 25 DEGs revealed many biological processes associated with cardiac function and the development of cardiac hypertrophy. In conclusion, we identified important similarities between ET-1-stimulated human-stem-cell-derived cardiomyocytes and human hypertrophic cardiac tissue. Novel putative cardiac hypertrophy biomarkers were identified and validated on the protein level, lending support for further investigations to assess their potential for future clinical applications.
