**3. MicroRNAs as autocrine, paracrine and endocrine molecules**

Region 8, **Figure 2A** [34]. This cleavage by DROSHA/DGCR8 produces a 60 nucleotide stemloop structure with a 3′ overhang, the pre-miRNA [11, 34, 35]. The primary miRNA can also be further subjected to RNA editing by ADARs (adenosine deaminases acting on RNA) that

**Figure 2.** MicroRNA biogenesis and function. (A) Primary miRNAs are cleaved in the nucleus by the RNase III endoribonuclease DROSHA and DGCR8. (B) Once the primary miRNA is cleaved, the nuclear transport receptor exportin 5 binds the 3′ overhang structure of the pre-miRNA to export it to the cytoplasm. (C) The RNase III enzyme Dicer and TRBP and PACT target the pre-miRNA through the 3′ overhang, converting it into mature miRNA, liberating a duplex nucleotide structure with two nucleotides protruding at the 3′ end. (D) The guide strand is loaded into the RNAinduced silencing complex (RISC), and the passenger strand is degraded by RNases. (E) Complementary pairing with the seed region to mRNAs determines target binding and guides argonaute proteins to stop translation. Accumulation of untranslated mRNA in the cytoplasm allows recruitment of members of the GW182 protein family. (F) Deadenylase

Exportin 5 allows export of the pre-miRNA to the cytoplasm, **Figure 2B** [36], where Dicer and substrate stabilizing binding partners, TRBP (trans-activation response RNA-binding protein) and PACT (protein activator of RNA-activated protein kinase) facilitate conversion into mature miRNA, **Figure 2C** [12, 14]. Two strands result from the unwinding of the duplex, the guide (3p) and passenger (5p) strands. Most of miRNA effects are mediated by the 3′ form; the 5′ form comprises <10% of all miRNA reads in humans [36]. The guide strand is loaded into the RNA-induced silencing complex (RISC) and the passenger strand is degraded by RNases, **Figure 2D**. IsomiRs can also be produced at this step by trimming and capping of the mature

modify adenosine to inosine producing miRNA isoforms called isomiRs [36].

complexes cause destabilization of the transcript and further degradation by RNase activity.

176 Renin-Angiotensin System - Past, Present and Future

miRNA.

miRNAs not only shape the intracellular proteome within specific cell types in response to microenvironment stimuli and cues, but can also mediate intercellular effects by means of nanotubes, exosomes and binding proteins, all mechanisms of intercellular communication [49]. Moreover, extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, also participate in paracrine and endocrine signalling, as well as an intercellular transfer of miRNAs [50–52]. Exosomes, in particular, which are nanovesicles derived from endosomes are involved in cell-to-cell communication [53], contain significant amounts of miRNAs and are resistant to changes in temperature, pH and the effect of RNases making them reliable sources for screening [51, 54, 55]. miRNAs are transported by RNA-binding proteins and are taken up into intraluminal vesicles during the formation of multivesicular bodies in endosomes [56]. Upon fusion of the endosome to the plasma membrane, the intraluminal vesicles are released as exosomes and due to their lipid composition and size, they can easily transfer genetic material across lipid membranes [55, 56]. Several miRNAs are transferred in vivo and in vitro between fibroblasts, cardiomyocytes, human umbilical endothelial cells, mesenchymal stem cells, cardiac and cerebral endothelial cells [57, 58], while atheroprotective communication has been found between endothelial and smooth muscle cells through miRNAs [59].

miRNA transfer both propagates deleterious effects and helps recover cells from insults and prevent apoptosis. For example, miR-133 is increased in people with cardiovascular disease and is transferred through exosomes from multipotent mesenchymal stromal cells to astrocytes and neurons that promote recovery after stroke [60–63]. Furthermore, remote ischaemic conditioning, a technique of small cycles of ischaemia/reperfusion in distal extremities, was protective for cardiac and cerebrovascular effects in animal experiments and human clinical trials, with effects mediated by miRNAs such as miR-1 [64–69].

Exosomal circulating miRNAs have many properties that arguably make them ideal biomarkers, including their presence in peripheral blood, detection in many biological fluids, their stability in RNase-rich body fluids and their tissue-specific expression patterns. These have been described in cardio-cerebrovascular disorders, diabetes, dyslipidemia and neurodegenerative disorders [1, 70–76]. Furthermore, human exosomes can be used therapeutically as a gene delivery vector to provide cells with heterologous miRNAs [53].
