Stem cell-derived extracellular vesicles reduce the expression of molecules involved in cardiac hypertrophy-In a model of human-induced pluripotent stem cell-derived cardiomyocytes.

Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-ß1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-ß1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-ß1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.

Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players.

Atherosclerosis is a progressive, chronic inflammatory disease of the large arteries caused by the constant accumulation of cholesterol, followed by endothelial dysfunction and vascular inflammation. We hypothesized that delivery of extracellular vesicles (EVs), recognized for their potential as therapeutic targets and tools, could restore vascular function in atherosclerosis. We explored by comparison the potential beneficial effects of EVs from subcutaneous adipose tissue stem cells (EVs (ADSCs)) or bone marrow mesenchymal stem cells (EVs (MSCs)) on the consequences of atherogenic diet on vascular health. Also, the influences of siRNA-targeting Smad2/3 (Smad2/3siRNA) on endothelial dysfunction and its key molecular players were analyzed. For this study, an animal model of atherosclerosis (HH) was transplanted with EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3siRNA. For controls, healthy or HH animals were used. The results indicated that by comparison with the HH group, the treatment with EVs(ADSCs) or EVs(MSCs) alone or in combination with Smad2/3siRNA of HH animals induced a significant decrease in the main plasma parameters and a noticeable improvement in the structure and function of the thoracic aorta and carotid artery along with a decrease in the selected molecular and cellular targets mediating their changes in atherosclerosis: 1) a decrease in expression of structural and inflammatory markers COL1A1, α-SMA, Cx43, VCAM-1, and MMP-2; 2) a slight infiltration of total/M1 macrophages and T-cells; 3) a reduced level of cytosolic ROS production; 4) a significant diminution in plasma concentrations of TGF-ß1 and Ang II proteins; 5) significant structural and functional improvements (thinning of the arterial wall, increase of the inner diameter, enhanced distensibility, diminished VTI and Vel, and augmented contractile and relaxation responses); 6) a reduced protein expression profile of Smad2/3, ATF-2, and NF-kBp50/p65 and a significant decrease in the expression levels of miR-21, miR-29a, miR-192, miR-200b, miR-210, and miR-146a. We can conclude that 1) stem cell-derived EV therapies, especially the EVs (ADSCs) led to regression of structural and functional changes in the vascular wall and of key orchestrator expression in the atherosclerosis-induced endothelial dysfunction; 2) transfection of EVs with Smad2/3siRNA amplified the ability of EVs(ADSCs) or EVs(MSCs) to regress the inflammation-mediated atherosclerotic process.

Extracellular vesicles-incorporated microRNA signature as biomarker and diagnosis of prediabetes state and its complications.

Extracellular vesicles (EVs) are small anuclear vesicles, delimited by a lipid bilayer, released by almost all cell types, carrying functionally active biological molecules that can be transferred to the neighbouring or distant cells, inducing phenotypical and functional changes, relevant in various physio-pathological conditions. The microRNAs are the most significant active components transported by EVs, with crucial role in intercellular communication and significant effects on recipient cells. They may also server as novel valuable biomarkers for the diagnosis of metabolic disorders. Moreover, EVs are supposed to mediate type 2 diabetes mellitus (T2DM) risk and its progress. The T2DM development is preceded by prediabetes, a state that is associated with early forms of nephropathy and neuropathy, chronic kidney disease, diabetic retinopathy, and increased risk of macrovascular disease. Although the interest of scientists was focused not only on the pathogenesis of diabetes, but also on the early diagnosis, little is known about EVs-incorporated microRNA involvement in prediabetes state and its microvascular and macrovascular complications. Here, we survey the biogenesis, classification, content, biological functions and the most popular primary isolation methods of EVs, review the EVs-associated microRNA profiling connexion with early stages of diabetes and discuss the role of EVs containing specific microRNAs in prediabetes complications.

An in vitro-based hazard assessment of liquid smoke food flavourings.

Liquid smoke products are widely used as a food additive to create a desired smoke flavour. These products may contain hazardous chemicals generated during the wood-burning process. However, the toxic effects of these types of hazardous chemicals constituting in the commercially available products are largely unknown. Therefore, a test battery of cell-based in vitro methods, covering different modes of actions of high relevance to human health, was applied to study liquid smoke products. Ten liquid smoke flavourings were tested as non-extracted and extracted. To assess the potential drivers of toxicity, we used two different solvents. The battery of in vitro methods covered estrogenicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity and genotoxicity. The non-extracted samples were tested at concentrations 0.002 to 1 µL liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 µL/mL. Genotoxicity was observed for nearly all non-extracted and all hexane-extracted samples, in which the former had higher potency. No genotoxicity was observed for ethyl acetate-extracted samples. Oxidative stress was activated by almost all extracted and non-extracted samples, while approximately half of the samples had aryl hydrocarbon receptor and estrogen receptor activities. This study used effect-based methods to evaluate the complex mixtures of liquid smoke flavourings. The increased bioactivities seen upon extractions indicate that non-polar chemicals are driving the genotoxicity, while polar substances are increasing oxidative stress and cytotoxic responses. The differences in responses indicate that non-extracted products contain chemicals that are able to antagonize toxic effects, and upon extraction, the protective substances are lost.

Hypertension Associated With Hyperlipidemia Induced Different MicroRNA Expression Profiles in Plasma, Platelets, and Platelet-Derived Microvesicles; Effects of Endothelial Progenitor Cell Therapy.

Aim: The aim of this study was to analyze the expressed profiles of miRNAs in plasma, platelets, and platelet-derived microvesicles (PMVs) obtained from experimental induced atherosclerosis animal model and to investigate the effect of EPC transplantation on these profiles. Methods: Seventeen selected circulating miRNAs (miR-19a,-21,-126,-146a,-223,-26b,-92a,-222,-210,-221,-143,-10a,-145,-155,-34a,-204, and miR-214) were individually analyzed in plasma, platelets, and PMVs isolated from peripheral blood of hypertensive-hyperlipidemic hamsters treated or not with endothelial progenitor cells (EPCs), and of healthy hamsters taken as control group. Results: Comparative with control group, in hypertension associated with hyperlipidemia the investigated miRNA expression profiles were different: (i) in plasma, the levels of all investigated miRNAs were significantly increased, the highest enhances being noticed for miR-21,-146a,-221,-143,-34a, and miR-204; (ii) in platelets, the expressions of almost all miRNAs were significantly elevated, remarkable for miR-126,-146a,-223,-222, and miR-214, while levels of miR-143, miR-10a, and miR-145 were significantly reduced; (iii) in PMVs, numerous miRNAs were found to have significantly increased levels, especially miR-222,-221,-210, and miR-34a, whereas expressions of various miRNAs as miR-223,-214,-146a,-143,-10a, and miR-145 were significantly decreased. The treatment with EPCs had the following reverse effects: (i) in plasma, significantly reduced the expression of miR-26b,-143,-34a,-204, and miR-214; (ii) in platelets, significantly decreased the levels of almost investigated miRNAs, with remarkably diminishing for miR-126 and miR-221; and (iii) in PMVs, significantly lowered the expression of some miRNAs, with considerably reductions for miR-222,-221,-210, and miR-19a, while the level of miR-214 was found elevated. Conclusions: The present study revealed that miRNAs have differential expression profiles in plasma, platelets, and PMVs under hypertension associated with hyperlipidemia conditions. The different miRNA profile in PMVs compared with platelets indicated an active mechanism of selective packing of miRNAs into PMVs from maternal cells; various miRNAs such as miR-19a,-21,-126,-26b,-92a,-155,-204,-210,-221,-222, and-34a delivered by PMVs may contribute to enrichment of circulating plasma miRNA expression. In addition, our study showed that the EPC-based therapy can regulate the expressions of investigated miRNAs into the three sources. These results provide novel information that could help in finding potential targets for the development of new therapeutic strategies in the cardiovascular disease.