Friday, April 26, 2024
4/26/2024
PROJECT SUMMARY Phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative phenotype, plays a causal role in many human occlusive vascular diseases. However, the key factors critical for the event are far from completely identified. Emerging evidence suggests that long non-coding RNAs (lncRNAs) are critical for gene expression but VSMC-specific lncRNAs remain ill-defined. In an effort to identify lncRNAs with a role in regulating VSMC phenotype, we utilized publicly available RNA-seq and ChIP-seq data sets that are generated from different cells/tissues to identify VSMC-enriched lncRNAs. This unbiased analysis revealed that the lncRNA CARMN is specifically expressed in VSMCs. Correlation analysis revealed that SM-specific expression of CARMN is not only correlated with a set of well-known SM-contractile markers, but also with serum response factor (SRF) and its cardiac/SM-specific cofactor myocardin (MYOCD). SRF/MYOCD is a transcriptional complex that plays a critical role in regulating SM-specific contractile gene expression, through binding to the CArG boxes within these genes. Bioinformatic analysis identified 2 evolutionarily conserved CArG boxes within CARMN gene locus and our exciting preliminary data further demonstrated that CARMN expression is SRF/MYOCD- dependent. Furthermore, we found that CARMN is down-regulated during VSMC phenotypic switching in vivo and in vitro. More importantly, depletion of CARMN inhibits while overexpression of CARMN promotes the contractile phenotype of VSMCs. Remarkably, our exciting preliminary data further showed that CARMN acts as a transcriptional activator by binding to MYOCD but also synergistically enhancing MYOCD-mediated transactivation on SM-specific genes including CARMN itself. Therefore, we hypothesize that SRF/MYOCD drives SM-specific lncRNA CARMN expression and that CARMN plays a critical role in maintaining the contractile state of VSMCs through a positive feedback mechanism by binding to MYOCD. Three aims are proposed to test this novel hypothesis. In Aim 1, we will determine the regulatory mechanism by which CARMN specifically expresses in VSMCs by using our novel CARMN knock-in reporter mice, ChIP assay, mutagenesis in vitro and CRISPR-Cas9 genome editing of CArG box in vivo. In Aim 2, we will define the functional role of CARMN in VSMCs by using our novel inducible SM-specific CARMN KO mice and rat carotid artery balloon injury model. In Aim 3, we will explore the mechanism of CARMN's action in VSMCs by assessing the physical and functional binding of CARMN with MYOCD. Completion of these studies will provide novel insights into the mechanisms controlling VSMC phenotypic plasticity and identify the novel SM-specific lncRNA CARMN for treating many proliferative vascular diseases.
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