Role of an enhancer transcribed long non‐coding RNA Alive in the transdifferentiation of Vascular Smooth Muscle Cells to Chondrocytes in response to Angiotensin II
Authors: AMARAM VISHNU , DAS SADHAN , LEUNG AMY , REDDY MARPADGA A. , BANSAL ANITA , LANTING LINDA , NATARAJAN RAMA 
Published: June 2021

Vascular Smooth Muscle Cells (VSMC) provide structural integrity to the vessel wall and have contractile responses to various vasoactive stimuli. Angiotensin II (AngII), a peptide hormone binds to a G‐protein coupled receptor (AT1R), is a pro‐inflammatory and growth‐promoting factor involved in the development of hypertension and atherosclerosis by promoting VSMC dysfunction. Long noncoding RNAs (lncRNAs) are emerging as important regulators of gene expression and are associated with various disease processes. LncRNAs possess structural similarity to mRNAs, but do not possess coding potential. Recent studies show that enhancer genomic elements can be transcribed into a class lncRNAs that include enhancer lncRNAs. Using RNA‐ and ChIP‐sequencing, our lab identified several novel lncRNAs that are differentially regulated by Ang II in rat VSMC. Some of these are transcribed from Histone H3 lysine 27 acetylation (H3K27Ac) chromatin modification associated enhancer elements. In the current study, we examined the regulation and functions of “AngII induced lncRNA in vascular cells with enhancer function (Alive)”, a novel H3K27Ac enhancer transcribed lncRNA located 117kb upstream of a protein coding gene Aggrecan (Acan). Alive and its “nearby” gene Acan expressions were upregulated by AngII in time dependent manner in VSMCs as identified by RNA‐seq and validated by qRT‐PCR analyses. We identified increased enrichments of H3K27Ac and RNA pol II S2P binding in the Alive genomic locus after AngII treatment suggesting Alive is transcribed from an active enhancer. Acan codes for an extra cellular matrix protein that is expressed in chondrocytes and helps in the maintenance of cartilage. Acan is expressed in VSMCs only in response to vascular injury or pathologic stimuli such as AngII, platelet derived growth factor (PDGF) and is associated with cardiovascular diseases (CVD). It has been shown previously that VSMCs undergo transdifferentiation to chondrocytes in a mouse model of arterial injury. We found that Alive and Acan expressions in VSMCs are also similarly upregulated by PDGF. Subcellular fractionation and RT‐qPCRs, as well as RNA‐FISH analyses showed that Alive is predominantly localized in the nucleus. Functional analysis revealed that knockdown (KD) of Alive with locked nucleic acid (LNA)‐GapmeRs in rat VSMC also decreased the expression of nearby gene Acan. Microarray profiling, followed by gene ontology analysis of Alive KD cells revealed a role for Alive in Cell‐to‐Cell signaling, and Cell movement functions. Furthermore, KD of Alive decreased AngII induced expression of genes such as Sox9, Runx1 that are key transcription factors associated with chondrocyte formation. We also identified Sox9 binding sites upstream of Acan suggesting a role for Alive and Sox9 in inducing Acan expression in response to AngII. Overall, our results suggest Alive is an AngII induced enhancer associated lncRNA that may mediate the transdifferentiation of VSMCs to chondrocytes to promote vascular dysfunction and CVD.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.