**3. MicroRNAs**

MicroRNAs are endogenous, small non-coding RNA molecules that inhibit translation of mRNAs. The microRNA's target selection is predominantly determined by the microRNA's 'seed sequence', nucleotides 2–8 at the 5--end of a microRNA [25,26], which bind their target mRNAs via Watson–Crick base-pairing. Due to this relatively small targeting sequence, a single microRNA's 'targetome' can consist of hundreds of mRNAs, enabling microRNAs to regulate multifactorial processes [10].

The biogenesis of microRNAs starts with the transcription of the microRNA containing gene, yielding a primary microRNA (pri-miR) which then undergoes several steps of maturation to form the mature and functional microRNA (Figure 1) [27]. First, the pri-miR is cleaved in the nucleus by Drosha to generate a hairpin-shaped precursor microRNA (pre-miR) [28]. The pre-miR is then translocated to the cytoplasm where a final cleavage is performed by Dicer, yielding a microRNA duplex [29]. Either side of the duplex can associate with Argonaute proteins and become a functional mature microRNA after incorporation into the RNA-induced silencing complex (RISC) [30]. Mature microRNAs are named after their side, 5- or 3-, in the pri-miR hairpin (e.g., miR-#-5p or -3p).

**Figure 1.** MicroRNA biogenesis and alterations that induce isomiR formation or microRNA nucleotide modifications. Transcription of the microRNA containing gene forms the primary microRNA (pri-miR). Drosha cleaves the pri-miR to generate the precursor microRNA (pre-miR). The pre-miR cleaved by Dicer in the cytoplasm yielding the microRNA duplex. Either side of the duplex can be incorporated into the RNA-induced silencing complex (RISC) to become a functional mature microRNA. IsomiRs can be formed during microRNA biogenesis when Drosha or Dicer cleave in alternative locations, or when exonucleases or nucleotidyl transferases remove or add nucleotides to the 3--end of the pre-miR or the mature microRNA. RNA nucleotide modifications with known or potential functional implications on microRNA biogenesis or functioning are shown in red with their 'writers' next to them.

MicroRNA biogenesis is strictly regulated, even at a microRNA-specific level, by numerous factors, including DNA methylation, activity modulation of key maturation proteins and many RNA-binding proteins [29,31–33]. As a result, microRNA expression is often highly tissue specific and is dynamically regulated during key physiological processes, including the response to ischemia [29,34].

In 2007, the importance of microRNAs in neovascularization was demonstrated for the first time when several studies showed that Dicer-dependent microRNAs were required for angiogenesis [35–37]. Since then, microRNAs have been shown to play a functional role in all processes involved in neovascularization, including production and secretion of angiogenic stimuli, as well as EC, SMC, fibroblast and immune cell proliferation, migration and activation, which have recently been reviewed in references [8,38–40]. Several of these vasoactive microRNAs have also been well described to play an important role in vascular remodeling during ischemic cardiovascular diseases [8,41].

For example, Bonauer et al. showed that miR-92a is highly expressed in human ECs and functions as negative regulator of angiogenesis [42]. Inhibition of miR-92a increased angiogenesis in vivo and improved blood flow recovery after hindlimb ischemia [42]. Furthermore, administration of miR-92a inhibitors in porcine models for myocardial infarction demonstrated that miR-92 inhibition prevents adverse infarct remodeling and ischemia/reperfusion injury [43,44]. Phase 1 trials aimed to improve wound healing with a future potential clinical application towards heart failure treatment have recently been completed for miR-92a inhibitor MRG 110 (NCT03603431) [45].

Both miR-126-3p and -5p are also highly expressed in ECs where they promote angiogenesis by stimulating EC proliferation and VEGF signaling and regulating leukocyte adhesion [46–49]. Inhibition of miR-126-3p was shown to decrease recovery after myocardial infarction and hindlimb ischemia in mice [47,50,51]. Furthermore, miR-126 levels are decreased in patients with ischemic coronary artery disease [52]. Similarly, miR-10a also stimulates angiogenesis by promoting VEGF signaling in ECs and regulating their inflammatory phenotype [53–57].

MiR-21-5p regulates proliferation and apoptosis of vascular wall smooth muscle cells [58,59] and promotes fibrosis by stimulating fibroblast survival and growth factor secretion [60]. Preclinical studies have shown that inhibition of miR-21-5p can prevent maladaptive vascular remodeling and heart failure [59,60]. These findings sugges<sup>t</sup> that the miR-21-5p inhibitor RG-012, which is currently being tested in a phase 2 clinical trial to prevent kidney fibrosis in patients with Alport syndrome (NCT02855268), could potentially be used for the treatment of CVD.

Additionally, it is noteworthy that several groups of genomically clustered microRNAs have been identified that are able to broadly regulate neovascularization in response to ischemia: Knockout of the miR-17/92 gene cluster (located on chromosome 14 in mice and on human chromosome 13) increased both angiogenesis and arteriogenesis [61,62], while the inhibition of individual microRNAs from the 14q32 microRNA cluster (located on chromosome 12F1 in mice and on human chromosome 14) was shown to independently stimulate both angiogenesis and arteriogenesis [9].

#### **4. IsomiRs and the microRNA Epitranscriptome**

Typically, microRNAs have been defined as a single sequence of RNA nucleotides, and are listed as such in the principle public microRNA database, miRbase [63]. However, recent studies have shown that this 'canonical' microRNA sequence can be altered. These microRNA alterations can be separated into two types: isomiRs and RNA nucleotide modifications.

IsomiRs are microRNA sequence variants that have one or more nucleotides added or deleted at their 5-- and/or 3--ends compared to the canonical microRNA sequence.

RNA nucleotide modifications are biochemical modifications of the standard RNA nucleotides, which are performed by enzymes present in all living organisms. Recent studies have demonstrated that these RNA nucleotide modifications have a functional regulatory role and form what has been named the 'epitranscriptome' [64]. While many di fferent RNA nucleotide modifications exist, only a few have been studied in the context of microRNAs: Adenosine-to-inosine editing (A-to-I editing) and N6-adenosine methylation (m6A) and 2- -*O*-methylation (2-OMe).

Below, we will discuss those studies that demonstrate that isomiRs and microRNA A-to-I editing and m6A can be actively regulated and play a directing role in neovascularization, as well as other modifications (including 2-OMe) that are likely to have a similar role.
