RESUMO
[Figure: see text].
Assuntos
Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/enzimologia , Isquemia/enzimologia , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Infarto do Miocárdio/enzimologia , Neovascularização Fisiológica , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Metiltransferases/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Transdução de Sinais , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMO
MicroRNAs (miRNAs) regulate gene expression by post-transcriptional inhibition of target genes. Proangiogenic small extracellular vesicles (sEVs; popularly identified with the name "exosomes") with a composite cargo of miRNAs are secreted by cultured stem cells and present in human biological fluids. Lipid nanoparticles (LNPs) represent an advanced platform for clinically approved delivery of RNA therapeutics. In this study, we aimed to (1) identify the miRNAs responsible for sEV-induced angiogenesis; (2) develop the prototype of bioinspired "artificial exosomes" (AEs) combining LNPs with a proangiogenic miRNA, and (3) validate the angiogenic potential of the bioinspired AEs. We previously reported that human sEVs from bone marrow (BM)-CD34+ cells and pericardial fluid (PF) are proangiogenic. Here, we have shown that sEVs secreted from saphenous vein pericytes and BM mesenchymal stem cells also promote angiogenesis. Analysis of miRNA datasets available in-house or datamined from GEO identified the let-7 family as common miRNA signature of the proangiogenic sEVs. LNPs with either hsa-let-7b-5p or cyanine 5 (Cy5)-conjugated Caenorhabditis elegans miR-39 (Cy5-cel-miR-39; control miRNA) were prepared using microfluidic micromixing. let-7b-5p-AEs did not cause toxicity and transferred functionally active let-7b-5p to recipient endothelial cells (ECs). let-7b-AEs also improved EC survival under hypoxia and angiogenesis in vitro and in vivo. Bioinspired proangiogenic AEs could be further developed into innovative nanomedicine products targeting ischemic diseases.
Assuntos
Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Lipossomos/química , MicroRNAs/metabolismo , Nanopartículas/química , Neovascularização Fisiológica , Líquido Pericárdico/fisiologia , Animais , Exossomos/genética , Vesículas Extracelulares/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Camundongos , MicroRNAs/genéticaRESUMO
N6-methyladenosine (m6A) is the most abundant and well-studied internal modification of messenger RNAs among the various RNA modifications in eukaryotic cells. Moreover, it is increasingly recognized to regulate non-coding RNAs. The dynamic and reversible nature of m6A is ensured by the precise and coordinated activity of specific proteins able to insert ("write"), bind ("read") or remove ("erase") the m6A modification from coding and non-coding RNA molecules. Mounting evidence suggests a pivotal role for m6A in prenatal and postnatal development and cardiovascular pathophysiology. In the present review we summarise and discuss the major functions played by m6A RNA methylation and its components particularly referring to the cardiovascular system. We present the methods used to study m6A and the most abundantly methylated RNA molecules. Finally, we highlight the possible involvement of the m6A mark in cardiovascular disease as well as the need for further studies to better describe the mechanisms of action and the potential therapeutic role of this RNA modification.
Assuntos
Adenosina/análogos & derivados , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/crescimento & desenvolvimento , Transcriptoma/genética , Adenosina/genética , Adenosina/metabolismo , Animais , Biomarcadores/metabolismo , Sistema Cardiovascular/metabolismo , Homeostase/genética , Humanos , Metilação , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismoRESUMO
MicroRNA-15a (miR-15a) and miR-16, which are transcribed from the miR-15a/miR-16-1 cluster, inhibit post-ischemic angiogenesis. MicroRNA (miRNA) binding to mRNA coding sequences (CDSs) is a newly emerging mechanism of gene expression regulation. We aimed to (1) identify new mediators of the anti-angiogenic action of miR-15a and -16, (2) develop an adenovirus (Ad)-based miR-15a/16 decoy system carrying a luciferase reporter (Luc) to both sense and inhibit miR-15a/16 activity, and (3) investigate Ad.Luc-Decoy-15a/16 therapeutic potential in a mouse limb ischemia (LI) model. LI increased miR-15a and -16 expression in mouse muscular endothelial cells (ECs). The miRNAs also increased in cultured human umbilical vein ECs (HUVECs) exposed to serum starvation, but not hypoxia. Using bioinformatic tools and luciferase activity assays, we characterized miR-15a and -16 binding to Tie2 CDS. In HUVECs, miR-15a or -16 overexpression reduced Tie2 at the protein, but not the mRNA, level. Conversely, miR-15a or -16 inhibition improved angiogenesis in a Tie2-dependent manner. Local Ad.Luc-Decoy-15a/16 delivery increased Tie2 levels in ischemic skeletal muscle and improved post-LI angiogenesis and perfusion recovery, with reduced toe necrosis. Bioluminescent imaging (in vivo imaging system [IVIS]) provided evidence that the Ad.Luc-Decoy-15a/16 system responds to miR-15a/16 increases. In conclusion, we have provided novel mechanistic evidence of the therapeutic potential of local miR-15a/16 inhibition in LI.