Extracellular vesicles from differentiated stem cells contain novel proangiogenic miRNAs and induce angiogenic responses at low doses.

Extracellular vesicles (EVs) released from healthy endothelial cells (ECs) have shown potential for promoting angiogenesis, but their therapeutic efficacy remains poorly understood. We have previously shown that transplantation of a human embryonic stem cell-derived endothelial cell product (hESC-ECP), promotes new vessel formation in acute ischemic disease in mice, likely via paracrine mechanism(s). Here, we demonstrated that EVs from hESC-ECPs (hESC-eEVs) significantly increased EC tube formation and wound closure in vitro at ultralow doses, whereas higher doses were ineffective. More important, EVs isolated from the mesodermal stage of the differentiation (hESC-mEVs) had no effect. Small RNA sequencing revealed that hESC-eEVs have a unique transcriptomic profile and are enriched in known proangiogenic microRNAs (miRNAs, miRs). Moreover, an in silico analysis identified three novel hESC-eEV-miRNAs with potential proangiogenic function. Differential expression analysis suggested that two of those, miR-4496 and miR-4691-5p, are highly enriched in hESC-eEVs. Overexpression of miR-4496 or miR-4691-5p resulted in increased EC tube formation and wound closure in vitro, validating the novel proangiogenic function of these miRNAs. In summary, we demonstrated that hESC-eEVs are potent inducers of EC angiogenic response at ultralow doses and contain a unique EV-associated miRNA repertoire, including miR-4496 and miR-4691-5p, with novel proangiogenic function.

Antisense MicroRNA Therapeutics in Cardiovascular Disease: Quo Vadis?

Heart failure (HF) is the end result of a diverse set of causes such as genetic cardiomyopathies, coronary artery disease, and hypertension and represents the primary cause of hospitalization in Europe. This serious clinical disorder is mostly associated with pathological remodeling of the myocardium, pump failure, and sudden death. While the survival of HF patients can be prolonged with conventional pharmacological therapies, the prognosis remains poor. New therapeutic modalities are thus needed that will target the underlying causes and not only the symptoms of the disease. Under chronic cardiac stress, small noncoding RNAs, in particular microRNAs, act as critical regulators of cardiac tissue remodeling and represent a new class of therapeutic targets in patients suffering from HF. Here, we focus on the potential use of microRNA inhibitors as a new treatment paradigm for HF.

Comparison of different chemically modified inhibitors of miR-199b in vivo.

MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3' to the 5' end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5' end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.