14q32 miRNA Cluster Takes Center Stage in Neovascularization

• Editorials
• angiogenesis effect
• endothelium
• microRNAs
• RNA, untranslated

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally control gene expression by binding to target mRNAs, thereby inducing degradation or translational inhibition. Various miRNAs regulate physiological and pathophysiological processes in the cardiovascular system and control angiogenesis and neovascularization as well as vessel remodeling and atherosclerosis.^1,2 miRNAs are expressed as primary transcripts that are subsequently processed by endonucleases into the mature forms. Primary transcripts often encode several miRs, so-called clusters. The 14q32 miRNA cluster is among the largest polycistronic clusters and comprises 54 miRNAs in humans and 61 in mice.³

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In this issue of Circulation Research, miRNAs of the 14q32 cluster were shown to regulate neovascularization after hindlimb ischemia.⁴ An interesting bioinformatics approach made the authors think that this miRNA cluster may be an important regulator of neovascularization. Authors selected genes that are known to control the complex process of neovascularization particularly angiogenesis and arteriogenesis and determined the enrichment of predicted miRNA target sites in these genes. Enrichment for target genes was observed for the previously described neovascularization-regulatory miRNAs, such as members of miR-17-92a and related clusters,⁵ and the miR-15a family.⁶ In addition, they identified putative target sites for 27 miRNAs from the 14q32 cluster. Silencing of 4 selected miRNAs, namely miR-329, miR-487b, miR-494, and miR-495, enhanced the recovery of blood flow after hindlimb ischemia, suggesting that at least these 14q32 cluster members impair neovascularization. Although the recovery of blood flow was similar after silencing of the different cluster members, targeting miR-329 and miR-487b showed a more profound effect on the remodeling of small to larger arteries and inhibition of miR-329, miR-487b, and miR-495 augmented capillary density. The effect of miR-487b on arterial remodeling may relate to the previously described repression of the antiapoptotic insulin receptor substrate 1, which is involved in remodeling of the aorta.⁷ Furthermore, the antiangiogenic effect of miR-329 is consistent with previous findings.⁸ The mechanisms by which miR-494 affects neovascularization, however, remained unclear because silencing of miR-494 did not significantly affect capillary density or arteriogenesis in vivo. This is even more surprising when taking into account that endothelial and myofibroblast proliferation and aortic outgrowth were profoundly stimulated by silencing of miR-494 in vitro. One may speculate that the observed increase in macrophage accumulation, an early endothelial protective effect or an influence on vasodilator functions, may underlie neovascularization improvement after miR-494 inhibition. Previous studies demonstrating that miR-494 targets both pro- and antiapoptotic genes in cardiomyocytes⁹ suggest that miR-494 may have a complex role in regulating tissue homeostasis.

Despite the impressive improvement of blood flow recovery achieved by silencing of the individual miRNAs, the authors subsequently struggled in defining the relevant target genes that are affected by the miRNAs. For example, in vitro luciferase assays showed that miR-329 directly repress the important proangiogenic transcription factor MEF2a; however, in vivo, Mef2a was only increased at day 7 after hindlimb ischemia but was significantly suppressed at day 3 after silencing of miR-329. Likewise, the demonstration of a direct repressing effect of miR-494 on FGFR2 (fibroblast growth factor receptor 2), VEGFA (vascular endothelial growth factor A), and EFNB2 (ephrin-B2) in luciferase activity assays was only reflected by a minimal derepression of the target genes after silencing of miR-494 in vivo. These discrepancies may in part relate to the sole measurement of mRNA expression in vivo, which precludes the detection of predominant translation inhibitory functions of miRNAs as well as the transient and modest target gene derepression mediated by miRNAs. Cell type–specific miRNA effects may also be overlooked when analyzing total tissue RNA. Despite the limited mechanistic insights into the detailed functions of the 14q32 miRNAs, the study provides many interesting aspects.

The study highlights a central role of the 14q32 cluster in vascular biology. The 14q32 miRNA cluster is well conserved in mammals and also regulated by imprinting. This imprinted genomic region is interesting as it comprises other noncoding RNAs such as the long noncoding RNA Meg3 and many C/D box small nucleolar RNAs, which primarily guide chemical modifications, particularly methylation, of other RNAs (Figure). Meg3 is increased by hypoxia in endothelial cells¹⁰ and in vivo deletion of Meg3, which also affects the expression of the 14q32 cluster, leads to increased vascularization of the brain.¹¹ Interestingly, VEGFA was found to be increased in Meg3-deficient mice,¹¹ a finding that is compatible with the targeting of VEGFA by several of the miRNAs that are encoded by the 14q32 cluster. It will be interesting to determine whether VEGFA derepression seen in Meg3^−/− mice is indeed related to deletion of Meg3 or is a consequence of the knockout of the 14q32 cluster miRNAs as one would predict from the study by Welten et al.⁴ The 14q32 genomic region has additional important functions in other pathological processes, such as oncogenesis, malignant growth, proliferation and cell survival, and cellular interactions with extracellular matrix, and it might play a role in the central nervous system.^12,13 Epigenetic mechanisms involved in imprinting and other potential regulatory functions of the 14q32 cluster certainly require further clarification. It is worth noting that some exogenous factors, such as cigarette smoke, have epigenetically mediated effects on the 14q32 cluster miRNAs during pulmonary carcinogenesis.¹⁴

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Figure.

Schematic illustration of the 14q22 locus. MicroRNAs (miRNAs) are enumerated according to the number and not chromosomal location. DMR indicates DNA methylated region; lncRNA, long noncoding RNA; and snoRNA, small nucleolar RNAs.

Of note, the miRNAs encoded by the 14q32 cluster showed a strikingly different expression pattern after limb ischemia.⁴ This is surprising because it had been suggested that all members of the 14q32 cluster are transcribed as 1 polycistronic RNA.³ However, Seitz et al³ explicitly state “… we cannot rule out that some miRNA genes are transcribed from their own promoter…”. Thus, the differential kinetics of the 14q32 cluster miRNAs may be either related to an unexplored transcriptional regulation of some miRNAs of the cluster or to hypoxia-regulated post-transcriptional regulation of miRNA processing. Indeed, the expression of other miRNA cluster members, for example, the miR-17-92a cluster, is also heterogeneous after ischemia and several pathways were shown to control the processing of miRNAs.¹⁵ For example, p53, which is induced by ischemia, was shown to affect processing of other miRNAs in endothelial cells.¹⁶

The study of Welten et al⁴ additionally opens up new potential targets for therapeutic applications for diseases related to problems in angiogenesis and beyond. Authors tested 2 different types of miRNAs inhibitors and showed that antagomirs and gene silencing oligonucleotide (GSOs)–based inhibitors are equally effective in vitro. Because antagomirs showed some toxicity in vitro, the authors used GSOs for the in vivo studies and showed that intravenously applied concentrations of 1 mg/mouse (which is ≈40 mg/kg) specifically and efficiently (with the exception of miR-495) inhibited the targeted miRNAs in control mice. The inhibition was more modest in ischemic legs where a significant inhibition was only detectable at some time points. Distribution studies further elegantly showed that GSO-based oligos are taken up by most organs except the brain, a finding that is consistent with the biodistribution of antagomirs and anti-miRs. Interestingly, the duration of inhibition was different from that observed before with antagomirs or LNA (locked nucleic acid)-based anti-miRs. Whereas a single injection of antagomirs and anti-miRs was reported to be effective for >4 weeks, GSO-mediated silencing was no longer detectable in most cases at day 17 with the exception of miR-487b targeting GSO, which showed a half maximal inhibition also at later time points.⁴ Although the use of miRNA inhibitors as therapeutic approach is clearly possible and warrants further studies, potential off-target effects need to be carefully addressed because miRNAs only need partial homology against their target mRNAs to mediate their effects. Moreover, in the present study, high doses were used systemically and the feasibility of using 40 mg/kg doses in humans (which would require 2–3 g GSOs in a 70 kg person) is unclear. In general, there is actually a need to develop sophisticated phenotypic screens and prediction programs to identify in vivo effects of nucleic acid–based therapeutics targeted toward miRNAs. In spite of these caveats, Welten et al⁴ report interesting findings that clearly deserve further studies and open up novel therapeutic opportunities.

• © 2014 American Heart Association, Inc.