A Novel Angiotensin II-Induced Long Noncoding RNA Giver Regulates Oxidative Stress, Inflammation, and Proliferation in Vascular Smooth Muscle Cells

Rationale: AngII (angiotensin II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long noncoding RNAs have elicited much interest, but their molecular roles in AngII actions and hypertension are unclear. Objective: To investigate the regulation and functions of a novel long noncoding RNA growth factor-and proinflammatory cytokine-induced vascular cell-expressed RNA (Giver), in AngII-mediated VSMC dysfunction. Methods and Results: RNA-sequencing and real-time quantitative polymerase chain reactions revealed that treatment of rat VSMC with AngII increased the expression of Giver and Nr4a3, an adjacent gene encoding a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with AngII. RNA-FISH (fluorescence in situ hybridization) and subcellular fractionation showed predominantly nuclear localization of Giver. AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and real-time quantitative polymerase chain reaction validation in VSMC showed that Giver knockdown attenuated the expression of genes involved in oxidative stress (Nox1) and inflammation (Il6, Ccl2, Tnf) but increased Nr4a3. Conversely, endogenous Giver overexpression showed opposite effects supporting its role in oxidative stress and inflammation. Chromatin immunoprecipitation assays showed Giver overexpression also increased Pol II (RNA polymerase II) enrichment and decreased repressive histone modification histone H3 trimethylation on lysine 27 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA-pulldown combined with mass spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins and corepressors, including NONO (non-pou domaincontaining octamer-binding protein). Moreover, NONO knockdown elicited similar effects as Giver knockdown on the expression of key Giver-regulated genes. Notably, GIVER and NR4A3 were increased in AngII-treated human VSMC and in arteries from hypertensive patients but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or angiotensin receptor blockers. Furthermore, human GIVER also exhibits partial functional conservation with rat Giver. Conclusions: Giver and its regulator Nr4a3 are important players in AngII-mediated VSMC dysfunction and could be novel targets for antihypertensive therapy.