In response to vascular injury, vascular smooth muscle cells (VSMCs) may switch from a contractile to a proliferative phenotype thereby contributing to neointima formation. Previous studies showed that the long noncoding RNA (lncRNA) is critical for paraspeckle formation and tumorigenesis by promoting cell proliferation and migration. However, the role of in VSMC phenotypic modulation is unknown. Herein we showed that expression was induced in VSMCs during phenotypic switching in vivo and in vitro. Silencing in VSMCs resulted in enhanced expression of SM-specific genes while attenuating VSMC proliferation and migration. Conversely, overexpression of in VSMCs had opposite effects. These in vitro findings were further supported by in vivo studies in which knockout mice exhibited significantly decreased neointima formation following vascular injury, due to attenuated VSMC proliferation. Mechanistic studies demonstrated that sequesters the key chromatin modifier WDR5 (WD Repeat Domain 5) from SM-specific gene loci, thereby initiating an epigenetic "off" state, resulting in down-regulation of SM-specific gene expression. Taken together, we demonstrated an unexpected role of the lncRNA in regulating phenotypic switching by repressing SM-contractile gene expression through an epigenetic regulatory mechanism. Our data suggest that is a therapeutic target for treating occlusive vascular diseases.

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Background: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of non-coding genes. Methods: We re-analyzed large-scale, publicly available bulk and scRNA-seq datasets from multiple tissues and cell types to identify VSMC-enriched lncRNAs. The in vivo expression pattern of a novel SMC expressed lncRNA, Carmn (CARdiac Mesoderm Enhancer-associated Non-coding RNA) was investigated using a novel Carmn GFP knock-in reporter mouse model. Bioinformatics and qRT-PCR analysis were employed to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro , functional assays were performed by knocking down CARMN with antisense oligonucleotides and over-expressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation and luciferase reporter assays. Results: We identified CARMN , which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific lncRNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145 . CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro . In vivo , SMC-specific deletion of Carmn significantly exacerbated, while overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs Conclusions: CARMN is an evolutionarily conserved SMC-specific lncRNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first non-coding RNA discovered to interact with myocardin.

Pioglitazone (PIO), a PPARγ agonist that improves glycemic control in type 2 diabetes through its insulin-sensitizing action, has been shown to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. Although PPARγ-dependent and PPARγ-independent mechanisms have been attributed to its vasoprotective effects, the signaling events associated with PIO action in VSMCs are not fully understood. To date, the likely intermediary role of AMP-activated protein kinase (AMPK) toward PIO inhibition of VSMC proliferation has not been examined. Using human aortic VSMCs, the present study demonstrates that PIO activates AMPK in a sustained manner thereby contributing in part to inhibition of key proliferative signaling events. In particular, PIO at 30 μM concentration activates AMPK to induce raptor phosphorylation, which diminishes PDGF-induced mTOR activity as evidenced by decreased phosphorylation of p70S6K, 4E-BP1, and S6 and increased accumulation of p27kip1, a cell cycle inhibitor. In addition, PIO inhibits the basal phosphorylation of ERK in VSMCs. Downregulation of endogenous AMPK by target-specific siRNA reveals an AMPK-independent effect for PIO inhibition of ERK, which contributes in part to diminutions in cyclin D1 expression and Rb phosphorylation and the suppression of VSMC proliferation. Furthermore, AMPK-dependent inhibition of mTOR/p70S6K and AMPK-independent inhibition of ERK signaling occur regardless of PPARγ expression/activation in VSMCs as evidenced by gene silencing and pharmacological inhibition of PPARγ. Strategies that utilize nanoparticle-mediated PIO delivery at the lesion site may limit restenosis after angioplasty without inducing PPARγ-mediated systemic adverse effects.