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.

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-β1 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.

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Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): CNCS-UEFISCDI Background 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 hypothesised that delivery of extracellular vesicles (EVs), recognized for their potential as therapeutic targets and tools, could restore vascular function in atherosclerosis. Purpose The goal was to evaluate vascular consequences of inflammation along with its key molecular and cellular players as intermediate endpoints for cardiovascular disease (CVD), and to explore ‘in vivo’ the potential beneficial effect of EVs from subcutaneous adipose tissue (EVs-ADSCs) or bone marrow mesenchymal stem cells (EVs-MSCs). Methods Our experimental animal model of atherosclerosis (hypertensive-hyperlipidemic hamster-HH) was treated with EVs-ADSCs or EVs-MSCs transfected or not with Smad2/3siRNA. For comparison, healthy animals (control group) or HH group transfected with Smad2/3siRNA alone were used. Results The results showed that compared with the control group, HH group displayed: (1) a marked increase of plasmatic parameter levels (cholesterol, LDL-cholesterol, triglycerides); (2) an amplification of expression of inflammatory markers with role in vascular dysfunction (COL1A, α-SMA, Cx43, VCAM-1, MMP-2); (3) a massive infiltration of total/M1 macrophages and T-cells into thoracic aorta and carotid artery; (4) an increased level of cytosolic ROS production; (5) a significant increase in plasma concentration of TGF-β1 and Ang II proteins; (6) significant structural (thickening of the arterial wall and reduction of the inner diameter) and functional changes (reduced distensibility, increased pulse wave velocity and velocity time integral, and diminished contractile responses to noradrenaline (NA) and reduced relaxation to acetylcholine (ACh)) in both thoracic aorta and carotid artery; (7) an altered protein expression profile (Smad2/3, ATF-2, NF-kBp50/p65) and a significant increase in the expression level of the miRNA panel (miR21, miR-29a, miR-192, miR-200b, miR-210, miR-146a) associated with inflammation-induced endothelial dysfunction. In addition, by comparison with the HH group, all treatment groups showed a significant decrease in the levels of plasma parameters, but also a noticeable improvement in the structure and function of the investigated blood vessels along with a decrease in the key molecules that modulate the inflammatory response through soluble mediators and cellular components. Conclusion We can conclude that the administration for 4 months of atherogenic diet activated key regulatory molecules in the atherosclerotic process, respectively TGF-β1, Ang II and ROS, modulating main orchestrators of inflammation-mediated atherosclerotic CVD progression. Also, EV-based treatment of inflammation, especially the EV-ADSC-based one, led to regression of arterial dysfunction and its key molecular players.

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