Keep calm and carry on: miR-1298 prevents up-regulation of Cx43 and secures a quiescent vascular smooth muscle cell.

Atherosclerosis constitutes the leading cause of cardiovascular disease resulting in ischaemia of dependent organs due to arterial occlusion. It affects not only heart and brain, but also limbs and other organs with fatal consequences for the individuum. The process invokes profound remodelling of the entire vascular wall driven by inflammation, which affects endothelial cells and promotes the activation of vascular smooth muscle cells (VSMCs). In healthy vessels, VSMCs retain a quiescent, contractile state but in response to environmental cues (chemical and mechanical) they adopt an activated, synthetic state characterized also by proliferation and migration. This remarkable plasticity is achieved through a substantial change in gene expression profile, which is likely governed by epigenetic and/or transcriptional control mechanisms. In addition to transcriptional regulation, non-coding, single-stranded, small RNAs (typically 22 nucleotides) contribute substantially to the phenotypic modulation of VSMCs through post-transcriptional regulation of gene expression (mRNA degradation or translational repression). These so-called microRNAs (miRNAs/miRs) promote or inhibit the switch of VSMCs to the synthetic state. For example, the miR-143/145 cluster supports a contractile state and was downregulated by vascular injury, implicating that its lack fosters the synthetic state. Conversely, other miRNAs (miR-21, miR-221/222) were found to promote the synthetic state, proliferation of VSMCs, and to be enhanced in vascular injury. Most of these effects were attributable to the regulation of the expression levels of transcription factors, thereby modifying a multitude of genes and modulating or achieving the phenotypic switch of VSMCs. Due to the tremendous interest in this field of cellular gene regulation, new members are continuously added to this list of miRNAs (miR-663, miR-34c, and miR-195) holding the promise of modulating vascular remodelling during atherosclerosis and neointima formation. Hu et al. added miR-1298 to this ever expanding list. However, this miRNA did not act via regulation of transcription factors, but directly by the post-transcriptional modulation of the expression of the gap junction forming protein connexin 43 (Cx43). The authors started from the observation that miR-1298 is expressed in healthy human vessels in VSMCs, but strongly down-regulated in atherosclerotic arteries. Interestingly, this down-regulation occurred at the transcriptional level as a consequence of methylation-mediated epigenetic silencing. They demonstrated a hypermethylation (by 100%) of CpG-rich fragments in the region upstream of the miR-1298 core sequence in diseased arteries, an effective miR-1298 up-regulation by the inhibition of DNA methyltransferase in vitro, and an inhibition of the miR-1298 promoter due to methylation using a reporter gene assay. The functional consequences were evaluated vigorously in cultured VSMCs: overexpression of miR-1298 inhibited PDGF-induced cell cycle progression into the S-phase and attenuated VSMC proliferation and migration, suggesting that miR-1298 prevents the switch to the synthetic phenotype. Conversely, blocking miR-1298 specifically revealed the opposite, i.e. an increased number of VSMCs in the S-phase, enhanced proliferation and migration (Figure 1). Mechanistically, the authors examined Cx43 as a target based on its complementary mRNA 3′-UTR sequence allowing miR-1298 to prevent Cx43 translation. Indeed, Cx43 protein levels (shown by immunostaining and western blot) were enhanced in human atherosclerotic arteries, and concomitantly assessed miR-1298 levels were reduced suggesting a negative regulation. An inhibitory effect of miR-1298 on Cx43 was verified in cultured VSMCs at the protein and mRNA level, pointing towards Cx43 mRNA as a direct target of miR-1298 through accelerated degradation. In fact, they identified specific binding sites on the Cx43 mRNA for miR-1298 that are decisive for the regulation using reporter gene assays. However, effects on Cx43 may still be an innocent bystander and further evidence was required. To provide this, the authors transfected VSMCs with Cx43 that is non-responsive to regulation by miR-1298 (lentiviral approach omitting the crucial miRbinding sites). This elegant approach would render other miR-1298 effects intact. As intended, miR-1298 was not able to inhibit Cx43 expression using the lentiviral approach. The overexpression of Cx43