Extracellular vesicles from dental pulp mesenchymal stem cells modulate macrophage phenotype during acute and chronic cardiac inflammation in athymic nude rats with myocardial infarction.

BACKGROUND/AIMS: Extracellular vesicles (EVs) derived from dental pulp mesenchymal stem cells (DP-MSCs) are a promising therapeutic option for the treatment of myocardial ischemia. The aim of this study is to determine whether MSC-EVs could promote a pro-resolving environment in the heart by modulating macrophage populations. METHODS: EVs derived from three independent biopsies of DP-MSCs (MSC-EVs) were isolated by tangential flow-filtration and size exclusion chromatography and were characterized by omics analyses. Biological processes associated with these molecules were analyzed using String and GeneCodis platforms. The immunomodulatory capacity of MSC-EVs to polarize macrophages towards a pro-resolving or M2-like phenotype was assessed by evaluating surface markers, cytokine production, and efferocytosis. The therapeutic potential of MSC-EVs was evaluated in an acute myocardial infarction (AMI) model in nude rats. Infarct size and the distribution of macrophage populations in the infarct area were evaluated 7 and 21 days after intramyocardial injection of MSC-EVs. RESULTS: Lipidomic, proteomic, and miRNA-seq analysis of MSC-EVs revealed their association with biological processes involved in tissue regeneration and regulation of the immune system, among others. MSC-EVs promoted the differentiation of pro-inflammatory macrophages towards a pro-resolving phenotype, as evidenced by increased expression of M2 markers and decreased secretion of pro-inflammatory cytokines. Administration of MSC-EVs in rats with AMI limited the extent of the infarcted area at 7 and 21 days post-infarction. MSC-EV treatment also reduced the number of pro-inflammatory macrophages within the infarct area, promoting the resolution of inflammation. CONCLUSION: EVs derived from DP-MSCs exhibited similar characteristics at the omics level irrespective of the biopsy from which they were derived. All MSC-EVs exerted effective pro-resolving responses in a rat model of AMI, indicating their potential as therapeutic agents for the treatment of inflammation associated with AMI.

Identification of omega-3 oxylipins in human milk-derived extracellular vesicles with pro-resolutive actions in gastrointestinal inflammation.

Introduction: Premature infants (PIs) are at risk of suffering necrotizing enterocolitis (NEC), and infants consuming human milk (HM) show a lower incidence than infants receiving formula. The composition of HM has been studied in depth, but the lipid content of HM-derived small extracellular vesicles (HM sEVs) remains unexplored. Identifying these molecules and their biological effects has potential for the treatment of intestinal disorders in PIs and could contribute to the development of HM-based fortified formulas. Methods: We isolated HM sEVs from HM samples and analyzed their oxylipin content using liquid chromatography coupled to mass spectrometry, which revealed the presence of anti-inflammatory oxylipins. We then examined the efficacy of a mixture of these oxylipins in combating inflammation and fibrosis, in vitro and in a murine model of inflammatory bowel disease (IBD). Results: HM-related sEVs contained higher concentrations of oxylipins derived from docosahexaenoic acid, an omega-3 fatty acid. Three anti-inflammatory oxylipins, 14-HDHA, 17-HDHA, and 19,20-DiHDPA (ω3 OXLP), demonstrated similar efficacy to HM sEVs in preventing cell injury, inducing re-epithelialization, mitigating fibrosis, and modulating immune responses. Both ω3 OXLP and HM sEVs effectively reduced inflammation in IBD-model mice, preventing colon shortening, infiltration of inflammatory cells and tissue fibrosis. Discussion: Incorporating this unique cocktail of oxylipins into fortified milk formulas might reduce the risk of NEC in PIs and also provide immunological and neurodevelopmental support.

Reliable assessment of carbon black nanomaterial of a variety of cell culture media for in vitro toxicity assays by asymmetrical flow field-flow fractionation.

Carbon black nanomaterial (CB-NM), as an industrial product with a large number of applications, poses a high risk of exposure, and its impact on health needs to be assessed. The most common testing platform for engineered (E)NMs is in vitro toxicity assessment, which requires prior ENM dispersion, stabilization, and characterization in cell culture media. Here, asymmetric flow field-flow fractionation (AF4) coupled to UV-Vis and dynamic light scattering (DLS) detectors in series was used for the study of CB dispersions in cell culture media, optimizing instrumental variables and working conditions. It was possible to disperse CB in a non-ionic surfactant aqueous solution due to the steric effect provided by surfactant molecules attached on the CB surface which prevented agglomeration. The protection provided by the surfactant or by culture media alone was insufficient to ensure good dispersion stability needed for carrying out in vitro toxicity studies. On the other hand, cell culture media in combination with the surfactant improved dispersion stability considerably, enabling the generation of shorter particles and a more favourable zeta potential magnitude, leading to greater stability due to electrostatic repulsion. It was demonstrated that the presence of amino acids in the culture media improved the monodisperse nature and stability of the CB dispersions, and resulted in a turn towards more negative zeta potential values when the pH was above the amino acid isoelectric point (IEP). Culture media used in real cell culture scenarios were also tested, and in vitro toxicity assays were developed optimizing the compatible amount of surfactant.

Isolation and Lipidomic Screening of Human Milk Extracellular Vesicles.

Extracellular vesicles (EVs) are secreted by cells and can be found in biological fluids (e.g., blood, saliva, urine, cerebrospinal fluid, and milk). EV isolation needs to be optimized carefully depending on the type of biofluid and tissue. Human milk (HM) is known to be a rich source of EVs, and they are thought to be partially responsible for the benefits associated with breastfeeding. Here, a workflow for the isolation and lipidomic analysis of HM-EVs is described. The procedure encompasses initial steps such as sample collection and storage, a detailed description for HM-EV isolation by multistage ultracentrifugation, metabolite extraction, and analysis by liquid chromatography coupled to mass spectrometry, as well as data analysis and curation.

HIF-Overexpression and Pro-Inflammatory Priming in Human Mesenchymal Stromal Cells Improves the Healing Properties of Extracellular Vesicles in Experimental Crohn's Disease.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have therapeutic potential in the treatment of several immune disorders, including ulcerative colitis, owing to their regenerative and immunosuppressive properties. We recently showed that MSCs engineered to overexpress hypoxia-inducible factor 1-alpha and telomerase (MSC-T-HIF) and conditioned with pro-inflammatory stimuli release EVs (EVMSC-T-HIFC) with potent immunomodulatory activity. We tested the efficacy of EVMSC-T-HIFC to repolarize M1 macrophages (Mφ1) to M2-like macrophages (Mφ2-like) by analyzing surface markers and cytokines and performing functional assays in co-culture, including efferocytosis and T-cell proliferation. We also studied the capacity of EVMSC-T-HIFC to dampen the inflammatory response of activated endothelium and modulate fibrosis. Finally, we tested the therapeutic capacity of EVMSC-T-HIFC in an acute colitis model. EVMSC-T-HIFc induced the repolarization of monocytes from Mφ1 to an Mφ2-like phenotype, which was accompanied by reduced inflammatory cytokine release. EVMSC-T-HIFc-treated Mφ1 had similar effects of immunosuppression on activated peripheral blood mononuclear cells (PBMC) as Mφ2, and reduced the adhesion of PBMCs to activated endothelium. EVMSC-T-HIFc also prevented myofibroblast differentiation of TGF-ß-treated fibroblasts. Finally, administration of EVMSC-T-HIFc promoted healing in a TNBS-induced mouse colitis model in terms of preserving colon length and intestinal mucosa architecture and altering the ratio of Mφ1/ Mφ2 infiltration. In conclusion, EVMSC-T-HIFC have effective anti-inflammatory properties, making them potential therapeutic agents in cell free-based therapies for the treatment of Crohn's disease and likely other immune-mediated inflammatory diseases.

miR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are an emerging alternative to cell-based therapies to treat many diseases. However, the complexity of producing homogeneous populations of EVs in sufficient amount hampers their clinical use. To address these limitations, we immortalized dental pulp-derived MSC using a human telomerase lentiviral vector and investigated the cardioprotective potential of a hypoxia-regulated EV-derived cargo microRNA, miR-4732-3p. We tested the compared the capacity of a synthetic miR-4732-3p mimic with EVs to confer protection to cardiomyocytes, fibroblasts and endothelial cells against oxygen-glucose deprivation (OGD). Results showed that OGD-induced cardiomyocytes treated with either EVs or miR-4732-3p showed prolonged spontaneous beating, lowered ROS levels, and less apoptosis. Transfection of the miR-4732-3p mimic was more effective than EVs in stimulating angiogenesis in vitro and in vivo and in reducing fibroblast differentiation upon transforming growth factor beta treatment. Finally, the miR-4732-3p mimic reduced scar tissue and preserved cardiac function when transplanted intramyocardially in infarcted nude rats. Overall, these results indicate that miR-4732-3p is regulated by hypoxia and exerts cardioprotective actions against ischemic insult, with potential application in cell-free-based therapeutic strategies.

HIF-1α and Pro-Inflammatory Signaling Improves the Immunomodulatory Activity of MSC-Derived Extracellular Vesicles.

Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.

Extracellular Vesicles Secreted by Hypoxic AC10 Cardiomyocytes Modulate Fibroblast Cell Motility.

Extracellular vesicles (EVs) are small membrane vesicles secreted by most cell types with important roles in cell-to-cell communication. To assess their relevance in the context of heart ischemia, EVs isolated from the AC10 ventricular cardiomyocyte cell line (CM-EVs), exposed to normoxia (Nx) or hypoxia (Hx), were incubated with fibroblasts (Fb) and endothelial cells (EC). CM-EVs were studied using electron microscopy, nanoparticle tracking analysis (NTA), western blotting and proteomic analysis. Results showed that EVs had a strong preference to be internalized by EC over fibroblasts, suggesting an active exosome-based communication mechanism between CM and EC in the heart. In Matrigel tube-formation assays, Hx CM-EVs were inferior to Nx CM-EVs in angiogenesis. By contrast, in a wound-healing assay, wound closure was faster in fibroblasts treated with Hx CM-EVs than with Nx CM-EVs, supporting a pro-fibrotic effect of Hx CM-EVs. Overall, these observations were consistent with the different protein cargoes detected by proteomic analysis under Nx and Hx conditions and the biological pathways identified. The paracrine crosstalk between CM-EVs, Fb, and EC in different physiological conditions could account for the contribution of CM-EVs to cardiac remodeling after an ischemic insult.