Novel Long Noncoding RNA, Macrophage Inflammation-Suppressing Transcript (MIST), Regulates Macrophage Activation During Obesity.

OBJECTIVE: Systemic low-grade inflammation associated with obesity and metabolic syndrome is a strong risk factor for the development of diabetes mellitus and associated cardiovascular complications. This inflammatory state is caused by release of proinflammatory cytokines by macrophages, especially in adipose tissue. Long noncoding RNAs regulate macrophage activation and inflammatory gene networks, but their role in macrophage dysfunction during diet-induced obesity has been largely unexplored. Approach and Results: We sequenced total RNA from peritoneal macrophages isolated from mice fed either high-fat diet or standard diet and performed de novo transcriptome assembly to identify novel differentially expressed mRNAs and long noncoding RNAs. A top candidate long noncoding RNA, macrophage inflammation-suppressing transcript (Mist), was downregulated in both peritoneal macrophages and adipose tissue macrophages from high-fat diet-fed mice. GapmeR-mediated Mist knockdown in vitro and in vivo upregulated expression of genes associated with immune response and inflammation and increased modified LDL (low-density lipoprotein) uptake in macrophages. Conversely, Mist overexpression decreased basal and LPS (lipopolysaccharide)-induced expression of inflammatory response genes and decreased modified LDL uptake. RNA-pull down coupled with mass spectrometry showed that Mist interacts with PARP1 (poly [ADP]-ribose polymerase-1). Disruption of this RNA-protein interaction increased PARP1 recruitment and chromatin PARylation at promoters of inflammatory genes, resulting in increased gene expression. Furthermore, human orthologous MIST was also downregulated by proinflammatory stimuli, and its expression in human adipose tissue macrophages inversely correlated with obesity and insulin resistance. CONCLUSIONS: Mist is a novel protective long noncoding RNA, and its loss during obesity contributes to metabolic dysfunction and proinflammatory phenotype of macrophages via epigenetic mechanisms.

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 domain-containing 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.

Diabetes Mellitus-Induced Long Noncoding RNA Dnm3os Regulates Macrophage Functions and Inflammation via Nuclear Mechanisms.

Objective- Macrophages play key roles in inflammation and diabetic vascular complications. Emerging evidence implicates long noncoding RNAs in inflammation, but their role in macrophage dysfunction associated with inflammatory diabetic complications is unclear and was therefore investigated in this study. Approach and Results- RNA-sequencing and real-time quantitative PCR demonstrated that a long noncoding RNA Dnm3os (dynamin 3 opposite strand) is upregulated in bone marrow-derived macrophages from type 2 diabetic db/db mice, diet-induced insulin-resistant mice, and diabetic ApoE-/- mice, as well as in monocytes from type 2 diabetic patients relative to controls. Diabetic conditions (high glucose and palmitic acid) induced Dnm3os in mouse and human macrophages. Promoter reporter analysis and chromatin immunoprecipitation assays demonstrated that diabetic conditions induce Dnm3os via NF-κB activation. RNA fluorescence in situ hybridization and real-time quantitative PCRs of subcellular fractions demonstrated nuclear localization and chromatin enrichment of Dnm3os in macrophages. Stable overexpression of Dnm3os in macrophages altered global histone modifications and upregulated inflammation and immune response genes and phagocytosis. Conversely, RNAi-mediated knockdown of Dnm3os attenuated these responses. RNA pull-down assays with macrophage nuclear lysates identified nucleolin and ILF-2 (interleukin enhancer-binding factor 2) as protein binding partners of Dnm3os, which was further confirmed by RNA fluorescence in situ hybridization immunofluorescence. Furthermore, nucleolin levels were decreased in diabetic conditions, and its knockdown enhanced Dnm3os-induced inflammatory gene expression and histone H3K9-acetylation at their promoters. Conclusions- These results demonstrate novel mechanisms involving upregulation of long noncoding RNA Dnm3os, disruption of its interaction with nucleolin, and epigenetic modifications at target genes that promote macrophage inflammatory phenotype in diabetes mellitus. The data could lead to long noncoding RNA-based therapies for inflammatory diabetes mellitus complications.

lncRNA DRAIR is downregulated in diabetic monocytes and modulates the inflammatory phenotype via epigenetic mechanisms.

Long noncoding RNAs (lncRNAs) are increasingly implicated in the pathology of diabetic complications. Here, we examined the role of lncRNAs in monocyte dysfunction and inflammation associated with human type 2 diabetes mellitus (T2D). RNA sequencing analysis of CD14+ monocytes from patients with T2D versus healthy controls revealed downregulation of antiinflammatory and antiproliferative genes, along with several lncRNAs, including a potentially novel divergent lncRNA diabetes regulated antiinflammatory RNA (DRAIR) and its nearby gene CPEB2. High glucose and palmitic acid downregulated DRAIR in cultured CD14+ monocytes, whereas antiinflammatory cytokines and monocyte-to-macrophage differentiation upregulated DRAIR via KLF4 transcription factor. DRAIR overexpression increased antiinflammatory and macrophage differentiation genes but inhibited proinflammatory genes. Conversely, DRAIR knockdown attenuated antiinflammatory genes, promoted inflammatory responses, and inhibited phagocytosis. DRAIR regulated target gene expression through interaction with chromatin, as well as inhibition of the repressive epigenetic mark H3K9me2 and its corresponding methyltransferase G9a. Mouse orthologous Drair and Cpeb2 were also downregulated in peritoneal macrophages from T2D db/db mice, and Drair knockdown in nondiabetic mice enhanced proinflammatory genes in macrophages. Thus, DRAIR modulates the inflammatory phenotype of monocytes/macrophages via epigenetic mechanisms, and its downregulation in T2D may promote chronic inflammation. Augmentation of endogenous lncRNAs like DRAIR could serve as novel antiinflammatory therapies for diabetic complications.

Linking diabetic vascular complications with LncRNAs.

Diabetes leads to markedly accelerated rates of many associated macrovascular complications like hypertension and atherosclerosis, and microvascular complications like nephropathy and retinopathy. High glucose, the hallmark of diabetes, drives changes in vascular and inflammatory cells that promote the development of these complications. Understanding the molecular processes involved in the development of diabetes and its debilitating complications can lead to much needed newer clinical therapies. Recently, long-noncoding RNAs (lncRNAs) have been shown to be important in the biology of vascular cells and there is growing evidence that lncRNAs are also involved in the cell biology relevant to diabetic vascular complications. In this review, we provide an overview of lncRNAs that function in vascular cells, and those that have been linked to diabetic complications.