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

Abstract

Rationale:

Angiotensin II (AngII)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long non-coding RNAs (lncRNAs) 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 lncRNA “Growth factor- and pro-Inflammatory cytokine-induced Vascular cell-Expressed RNA (Giver)”, in AngII-mediated VSMC dysfunction.

Methods and Results:

RNA-sequencing and RT-qPCRs 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 and subcellular fractionation showed predominantly nuclear localization of Giver. AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and RT-qPCR 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. ChIP assays showed Giver overexpression also increased RNA-polymerase II (Pol II) enrichment and decreased repressive histone modification H3K27me3 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA pull-down combined with mass-spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins, and corepressors including NONO. 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 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 anti-hypertensive therapy.

INTRODUCTION

Hypertension, a major contributor to global mortality, affects approximately 26% of the population worldwide, and is expected to rise to 30% by 2025.¹ The detrimental effects of high blood pressure (BP) are manifested through the development of cardio- and cerebro-vascular diseases, such as coronary heart disease and stroke.² The renin-angiotensin system (RAS) plays an important role in regulating BP in pathophysiological conditions. The primary effector of this system is angiotensin II (AngII), generated by angiotensin converting enzyme (ACE) in circulation and/or local vasculature. Under physiological conditions, AngII dynamically modulates fluid volume, sodium-potassium balance and vasoconstriction to maintain normal BP. Under pathological conditions, however, enhanced AngII production and AngII type 1 receptor (AT₁R) signaling promotes contraction and growth of vascular smooth muscle cells (VSMC), resulting in arterial wall remodeling and elevated BP.³

VSMC, the major components of the arterial wall, are responsible for maintaining vascular tone in response to hemodynamic changes and humoral stimulation. Dysfunction of VSMC, including increased oxidative stress, inflammation, migration, hyperplasia, and hypertrophy, all of which are induced by AngII and other growth factors, plays critical roles in the pathogenesis of hypertension, restenosis and atherosclerosis.^{3, 4}

NR4A3 (NOR1) belongs to the orphan nuclear receptor 4a (NR4A) subfamily which includes two other members NR4A1 (NUR77) and NR4A2 (NURR1). It is an important immediate early response gene and promotes VSMC proliferation by regulating key cell cycle-related genes⁵. NR4A members share a common structure consisting of two transactivation domains and one highly conserved DNA binding domain, but lack ligand binding domains. Thus, the function of these nuclear receptors are mostly regulated via changes in expression and posttranslational modifications.⁵ Once induced, NR4A members activate target gene transcription by binding as monomers or homodimers to a canonical DNA sequence, the nerve growth factor-induced protein B-responsive element (NBRE), consisting of an octanucleotide AAAGGTCA sequence. In particular, NR4A homodimers preferentially bind to NBRE-related sequence, an inverted repeat of AAAT(G/A)(C/T)CA.⁵ NR4A receptors are induced by diverse stimuli, including growth factors and inflammatory cytokines.^{5, 6} However, little is known about the regulatory effects of AngII on NR4A3 expression or its downstream signaling pathways.

In recent years, there is heightened interest in gene regulatory mechanisms mediated by non-coding RNAs (ncRNAs) and their connections to the pathogenesis of cardiovascular diseases (CVD).^7–11 The ncRNAs can be further divided into several subclasses, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Compared to miRNAs (18–22 nt) that have distinct roles in targeting mRNAs, lncRNAs (>200 nt) can act via multiple mechanisms to regulate genes associated with diverse biological processes.^7–11 Recent studies show that vascular cell expressed lncRNAs, such as SENCR, MALAT1, SMILR, MYOSLID, and Paupar, can regulate transcription of both closely (cis action) and remotely located (trans action) protein-coding genes, and have important functions in vascular endothelial cells and VSMC.^{8–10, 12–16} However, the detailed regulatory mechanisms and functional roles of lncRNAs in cardiovascular pathology are still not well understood.

We previously showed that miRNAs play key roles in AngII actions in VSMC.¹⁷ Furthermore, using RNA-sequencing (RNA-seq) and chromatin immunoprecipitation linked to high throughput sequencing (ChIP-seq), we identified, for the first time, 466 AngII-regulated lncRNAs (Lnc-Angs) in rat VSMC (RVSMC). Moreover, we demonstrated that one of the AngII-induced novel lncRNAs (Lnc-Ang362) functions as the host transcript for miR-221 and miR-222 to further augment RVSMC proliferation.¹³ Recently, we also showed functional role for lnc-Ang383 that overlaps with enhancers/super-enhancers in regulating AngII-induced genes in VSMC.¹⁸ However, the functions of other AngII-regulated novel lncRNAs are not known. Furthermore, lncRNAs are poorly conserved among different species¹⁹, and potential AngII-regulated human lncRNAs have not yet been characterized in human VSMC or arteries.

In the current study, we investigated the upstream regulatory mechanisms, downstream target genes, and functional roles of a novel AngII-induced lncRNA, Lnc-Ang164, in VSMC. This lncRNA was worthy of investigation because it is located adjacent to Nr4a3, whose function in AngII-mediated vascular pathology is not yet known. Notably, our results show a highly coordinated spatiotemporal interplay between Lnc-Ang164 and Nr4a3 in AngII-mediated signaling and VSMC dysfunction. Because Lnc-Ang164 was induced not only by AngII, but also other growth factors and cytokines, we renamed it as Growth factor- and pro-Inflammatory cytokine-induced Vascular cell-Expressed lncRNA (Giver). Interestingly, we found that human orthologs GIVER and NR4A3 were upregulated in human VSMC treated with AngII, and in mammary arteries from hypertensive patients versus normals, and were reduced in hypertensive patients treated with RAS inhibitors, suggesting GIVER could be a novel therapeutic target for hypertension.

METHODS

The authors declare that all data supporting the findings of this study are available within the article and its Online supplementary files.

Detailed Methods and Statistics are available in the Online Supplement. RVSMC were isolated from thoracic aortas of 12-week-old male Sprague-Dawley rats. Gene expression was analyzed from VSMC, rat and C57BL/6 mice aortas, and non-hypertensive and hypertensive patients’ samples. Details of PCR and ChIP-PCR primers are provided in Tables I-III in the Online Supplement. Results are presented as Means + SEM.

RESULTS

AngII induces Giver and Nr4a3 expression in a time-dependent manner.

Evidence shows that many lncRNAs can regulate expression of neighboring genes on the same chromosome through cis-acting mechanisms.^{20, 21} Therefore, we analyzed RNA-seq data from AngII treated RVSMC¹³ to identify differentially expressed lncRNAs located within ± 100 kb of AngII-induced coding genes. We found that a novel lncRNA, Lnc-Ang164, which we renamed as Giver, and a nearby orphan nuclear receptor coding gene Nr4a3, located ~99 kb from Giver on rat chromosome 5, were both significantly increased at 3h after AngII treatment (Figure 1A). H3K4me3 peaks depict start sites of these genes. To verify the regulation of Giver and nearby Nr4a3, RVSMC were treated with AngII for 1–6h and gene expression analyzed by RT-qPCR. Giver expression was significantly upregulated as early as 1h and peaked at 3h, while Nr4a3 expression peaked at 1h and remained elevated up to 6h after AngII treatment (Figure 1B-C).

Figure 1. Rat Giver and Nr4a3 expression is induced by AngII, mitogenic and pro-inflammatory factors in RVSMC.

(A), Normalized RNA-seq and H3K4me3 ChIP-seq tracks at Lnc-Ang164 (Giver) and Nr4a3 genomic loci. (B-O), Expression of Giver and Nr4a3 in response to indicated treatments in RVSMC using RT-qPCR. Delta Ct (Giver-Cypa) values are shown for Giver expression (B). Mean+SEM; n=3; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 using one-way ANOVA Dunnett’s multiple comparison tests.

We next examined the effects of other growth factors, cytokines and diabetic conditions (high glucose, HG) known to promote VSMC dysfunction. Both 20% fetal bovine serum (FBS) and 10ng/ml platelet-derived growth factor-BB (PDGF-BB) elicited marked induction of Nr4a3 expression as shown before²². Interestingly, they also induced significant increases in Giver expression (Figure 1D-G). 30mmol/L HG, 5ng/ml transforming growth factor-beta1 (TGF-β), 20ng/ml tumor necrosis factor-alpha (TNF-α) and 100 ng/ml lipopolysaccharide (LPS) also significantly induced both Giver and Nr4a3 with similar patterns (Figure 1H-O). However, 20 ng/ml IL-4 failed to induce Giver, and elicited only a moderate increase in Nr4a3 at 1h (Online Figure IA-B).

Together, these results demonstrate that Giver and its nearby gene Nr4a3 exhibit similar expression patterns in VSMC (suggesting co-regulation) in response to AngII and other stimuli implicated in the pathogenesis of CVD.

AngII induces Giver and Nr4a3 in cultured human vascular cells, and in rat and mice aortas.

To assess whether Giver is conserved across species in human and mouse, we first used the LiftOver tool²³ to identify transcripts with similar genomic organization on human (hg19) and mouse (mm9) reference genomes. Potential human (hGIVER) and mouse (mGiver) orthologs were located ~99 kb upstream of corresponding NR4A3 gene similar to the rat genome (Figure 1A and Online Figure IIA-B). Sequence comparison using NCBI BLAST tool²⁴ revealed that rat Giver shares 75% and 51% homology with predicted mouse and human genes respectively. Subsequent RT-qPCR analysis showed that GIVER and NR4A3 were significantly induced by AngII in human VSMC (HVSMC) (Figure 2A-B), similar to RVSMC.

Figure 2. AngII-regulated GIVER expression in human cells (in vitro) and rodent aortas treated ex vivo.

(A-J), Gene expression of GIVER and NR4A3 in response to AngII in HVSMC (A-B), HUVEC (C-D), THP-1 cells (E-F), and ex vivo in rat (G-H) and mouse aortas (I-J). Mean+SEM; n=3 (for A-F); n=5 rat aortas (for G-H); n=3–6 mouse aortas (for I-J); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 using one-way ANOVA Dunnett’s multiple comparison tests (for A-H) and unpaired two-tailed t-tests (for I-J).

To further clarify whether other vascular cells and monocytes can also contribute to the increase of NR4A3 and GIVER in aortas, we analyzed their expression in human umbilical vein endothelial cells (HUVEC) and THP-1 monocytes. AngII increased NR4A3 and GIVER RNA expression in HUVEC in a time-dependent fashion (Figure 2C-D). In contrast, although NR4A3 RNA was increased at 1h, AngII did not alter GIVER expression in THP-1 human monocytes (Figure 2E-F). Together, these results show that GIVER RNA is markedly increased by growth factors and pro-inflammatory cytokines in vascular cells, but not monocytes, supporting the nomenclature Growth factor- and pro-Inflammatory cytokine-induced Vascular cell-Expressed lncRNA (GIVER).

We next investigated the expression of Giver and Nr4a3 in aortas treated ex vivo with AngII. As shown in Figure 2G-H, both Giver and Nr4a3 expressions were increased in rat aortas 1h and 3h after AngII treatment respectively. AngII also increased both Giver and Nr4a3 transcripts at 1h and 3h after treatment in mouse aortas (Figure 2I-J). In addition, PDGF-BB treatment significantly increased the expression of Giver and Nr4a3 transcripts at 3h in mouse aortas (Online Figure III A-B). Overall, these results suggest co-regulation of Giver and Nr4a3 by AngII and PDGF also in ex vivo rodent models.

Nr4a3 mediates AngII-induced transactivation of Giver promoter via NBRE sequences.

Data in the previous sections suggested a close temporal relationship between the expression of Giver and Nr4a3 in response to a variety of mitogens and cytokines (Figure 1). Evidence shows that NR4A3 transactivates target gene promoters by binding to different canonical NBRE sequences.⁵ TRANSFAC and Jasper^{25, 26} motif analysis of the Giver promoter spanning −3026 to +1bp verified the presence of several NBRE motifs (Figure 3A). Hence, we hypothesized that Nr4a3 may regulate Giver transcription in RVSMC via promoter NBRE cis-elements. To evaluate whether Nr4a3 is required for AngII-induced Giver expression, we performed siRNA mediated knockdown or overexpression of Nr4a3. Transfection of RVSMC with siRNAs targeting Nr4a3 (siNr4a3) reduced Nr4a3 protein and RNA in both control and AngII treated cells relative to control siNTC oligonucleotides (Figure 3B-C, respectively). Notably, NR4a3 knockdown also significantly reduced AngII-induced Giver expression by ~30% (Figure 3D). Conversely, transfection of phNR4A3 vector expressing human NR4A3 (gift from Dr. Dennis Bruemmer, University of Pittsburgh) increased NR4A3 RNA and protein levels, and also enhanced Giver transcript expression (~1.5 fold) versus empty vector (Figure 3E-G).

Figure 3. Nr4a3 regulates Giver by binding to NBRE-elements on the Giver promoter.

(A), Schematic diagram of NBRE and NBRE-related motifs relative to Giver TSS. (B-D), Nr4a3 protein levels quantified by western blot (B), and RNA levels of Nr4a3 (C) and Giver (D) by RT-qPCR in RVSMC transfected with siRNA targeting NR4a3 (siNR4a3), or control (siNTC) and treated ± AngII for 3h. (E-F), Relative gene expression of human NR4A3 (E) and its protein (F) in RVSMC transfected with human NR4A3 expression vector (phNR4a3) and empty vector (pEV). (G), RT-qPCR of Giver in RVSMC transfected with phNR4a3 and pEV. (H-I), Relative luciferase activity of wild type (WT) Giver promoter constructs (H), and NBRE-site deletion mutant (MUT) (I), treated ± AngII. (J-K), Relative luciferase activity of −1581 WT and −1581 MUT Giver promoter in RVSMC transfected with siNR4a3 or siNTC and treated ± AngII (J), and with phNR4a3 or pEV (K). (L-M), ChIP assays showing Nr4a3 enrichment at NBRE binding site on Giver (L) and Skp2 (M) promoters in RVSMC transfected with phNR4A3 and pEV. Mean+SEM; n=2 (for B and F); n=3 (for C-D, E, G-H and L-M); n=6 (for I-J; 3 biological replicates with 2 technical replicates for each); n=5 (for K; 3 biological replicates with 2 technical replicates for 2 of them); *P<0.05; **P<0.005; ***P<0.001; ****P<0.0001 using one-way (for C-D), two-way ANOVA (for H-K) Tukey’s multiple comparison tests, and unpaired two-tailed t-tests (for E, G, L-M).

To further identify the specific NBRE binding sites involved (Figure 3A), we constructed luciferase reporter constructs driven by a series of Giver WT promoter fragments spanning from −3026, −1581, −1456, and −202 to +160 bp (Figure 3H). Transient transfections and subsequent luciferase assays showed that AngII transactivated the −3026 promoter, but the highest increase (~4 fold) in transcriptional activity was observed with the −1581 WT construct. TRANSFAC analysis of −3026 and −1581 promoter fragments revealed nine ZNF333 binding sites between −3026 and −1581. ZNF333 is a Zinc finger protein which contains KRAB domain associated with transcriptional repression. This might explain why the −3026 WT construct showed lower transcriptional activity compared to −1581 WT. Activity of −1581 WT was lost with the −1456 and −202 WT deletions (Figure 3H), suggesting the presence of important positive regulatory sites between −1581 and −1456 bp. Interestingly, an overlapping inverted AAATACCA repeat (−1527/−1515 bp) was found within this 125 bp (Online Figure IV). Accordingly, deletion of this potential NBRE-related motif in −1581-MUT construct significantly attenuated AngII response compared to −1581 WT (Figure 3I). Moreover, siNr4a3 significantly inhibited AngII-induced transcriptional activity of −1581 WT, but had no significant effect on −1581 MUT promoter (Figure 3J). Conversely, Nr4a3 overexpression mimicked AngII actions by increasing basal transcriptional activity of −1581 WT, which was significantly attenuated in 1581 MUT (Figure 3K). In addition, ChIP assays with Nr4a3 antibody revealed that Nr4a3 overexpression significantly increased Nr4a3 enrichment at the Giver promoter compared to control transfected cells (Figure 3L). Skp2 promoter served as positive control for Nr4a3 binding (Figure 3M).²⁷

Collectively, these data demonstrate that Nr4a3 mediates AngII-induced Giver transcription via NBRE-related site located 1515 bp upstream of Giver’s transcription start site (TSS).

Giver is nuclear enriched and negatively modulates NR4a3 expression.

Next, we employed Rapid amplification of cDNA ends (RACE) to construct full length Giver transcript. The 5’- and 3’-RACE showed Giver transcript is 1,225 bp long with three exons confirming our RNA-seq annotation (Online Figure VA-B). We also determined that Giver was polyadenylated by oligo-dT column purification and RT-qPCR (Online Figure VC-D). DNA sequencing revealed an alternative poly A+ signal (TATAAA; 1193/1198), 27 bp prior to the start of actual poly A+ sequence (Online Figure VE), indicating potential regulation by post-transcriptional mechanisms. We also verified that Giver does not have protein coding potential by performing in vitro transcription/translation assays (Online Figure VF).

Because sub-cellular localization of lncRNAs is closely associated with their functions, we next examined levels of Giver transcripts in nuclear and cytoplasmic fractions prepared from both untreated and AngII-treated (3h) RVSMC. Using U6 as nuclear marker and tRNAf-met as a cytoplasmic marker, we confirmed purity of sub-cellular fractions (Figure 4A-B). Furthermore, RT-qPCR showed that Giver transcript was highly enriched in the nuclear fractions with or without AngII stimulation (Figure 4C). We further confirmed nuclear localization by RNA-FISH after hybridization with probes targeting Giver (Figure 4D). These results suggest that Giver is polyadenylated and functions mainly in the nucleus. Having demonstrated that Giver is predominantly nuclear, we next examined its role in gene regulation. Because Nr4a3 is an AngII-regulated nearby gene, and Nr4a3 could also regulate Giver, we examined whether Giver also reciprocally regulates Nr4a3. We knocked down Giver with Dicer substrate siRNAs targeting Giver exon sequence (siGiver) in RVSMC. Compared to the non-targeting control siRNA (siNTC), Giver transcript was reduced by ~80% and ~50% in siGiver-transfected RVSMC with or without AngII treatment, respectively (Figure 4E). Surprisingly, Giver knock-down did not reduce Nr4a3 expression, but instead enhanced Nr4a3 expression in basal and AngII treated cells (Figure 4F), suggesting that AngII-induced Giver might suppress Nr4a3 expression, likely in a negative feedback regulatory loop.

Figure 4. Giver is nuclear enriched and regulates Nr4a3 expression through a negative feedback loop upon AngII signaling.

(A-C), RT-qPCR analysis of indicated RNAs from cytoplasmic (Cyto) and nuclear (Nuc) fractions from RVSMC treated ± AngII for 3h. (D), RNA-FISH analysis of Giver in RVSMC treated ± AngII or PDGF. Blue represents nuclear staining with DAPI. (E-F), RT-qPCR analysis of Giver (E), Nr4a3 (F) after siNTC or siGiver transfection ± AngII treatment. (G-H), RT-qPCR analysis of Giver (G) and Nr4a3 (H) 48h after transfection with empty vector (pEV) or rat Giver expression plasmid (pGiver). (I), Schematic depicting the location of sgRNAs targeting promoter (PR) and exons (EX) relative to Giver TSS for CRISPR-dCas9 mediated overexpression. (J-K), RT-qPCR analysis of Giver (J) and Nr4a3 (K) 48h after transfection with pAC154-dual-dCas9VP160-sg expression vectors expressing indicated sgRNAs. Mean+SEM; n=3 (for A-C); n=6 (for E-F and J-K); n=5 (for G-H); *P<0.05; ***P<0.001; ****P<0.0001; using one-way ANOVA Tukey’s (for A-C and E-F) and Dunnett’s (for J-K) multiple comparison tests, and unpaired two-tailed t-test (for G) and Mann-Whitney test (for H).

To further verify this negative effect of Giver on Nr4a3, we first cloned full-length Giver into pcDNA3.1 (+) vector (pGiver) (Online Figure VIA-B). Transient transfection of RVSMC with pGiver significantly increased Giver expression (Figure 4G). But, exogenously overexpressing Giver (ectopic) did not significantly reduce Nr4a3 expression, despite a trend towards decrease (Figure 4H). Furthermore, we also used the CRISPR-on method to overexpress endogenous Giver (Online Figure VIC). We designed two groups of sgRNAs, targeting either the promoter (PR) or exonic regions (EX) of Giver, and cloned these sgRNAs into pAC154-dual-dCas9VP160-sg vector. Of note, the target DNA sequences of PR sgRNAs were located within 200 bp upstream of TSS, while EX sgRNAs spanned 3 different exons (Figure 4I). As shown in Figure 4J, Giver expression was enhanced ~2 fold in PR group but not significantly altered in EX, compared to control vector (EV). In contrast, Nr4a3 was decreased in PR sgRNA-transfected RVSMC but unaffected in EX (Figure 4K). These results, suggest that Giver negatively modulates Nr4a3 expression at the transcriptional level.

We next examined whether Giver modulates other nearby genes besides Nr4a3 (−99 kb). The expression of five other genes within ±500 kb of Giver was not affected by Giver knockdown (Online Figure VID-I). Together, these data strongly suggest that AngII-induced Giver mainly suppresses Nr4a3, without affecting other proximal genes.

GIVER promotes inflammatory gene expression, oxidative stress, and proliferation in RVSMC.

In order to determine the putative functional roles of Giver in VSMC, we first performed unbiased microarray profiling of RVSMC transfected with siGiver to identify potential target genes. We found substantial differences in gene expression in RVSMC transfected with siGiver versus control siNTC (Online Figure VIIA-B) in untreated and AngII treated cells. We identified a total of 565 differentially expressed transcripts in untreated (257 down-regulated and 308 up-regulated) and 1214 transcripts in AngII-treated RVSMC (570 down-regulated and 644 up-regulated), respectively after Giver knockdown (FDR <0.005, log2 fold change ≥1.25). As expected, Nr4a3 was increased after rGiver knockdown in AngII treated cells. Interestingly, NADPH oxidase1 (Nox1) was among the top 10 downregulated genes in both untreated and AngII-treated RVSMC transfected with Giver siRNA (Online Table IV-VII). Moreover, pro-inflammatory genes, such as C-C motif chemokine ligand 2 (Ccl2) and Interleukin 6 (Il6), were also among the downregulated genes in both untreated and AngII-treated RVSMC (Online Figure VIIC-D). Furthermore, Ingenuity Pathway Analysis (IPA) revealed that inflammatory response, cell cycle, and cell growth were strongly associated with Giver knockdown (Online Figure VIIIA-D). These results indicate that Giver may regulate oxidative stress, inflammation, and proliferation in RVSMC.

Giver knockdown differentially regulated (up- and downregulated) the expression of genes not only on chr5 on which it is located, but also those present on other chromosomes (Figure 5A). Using RT-qPCR we validated that Giver knockdown significantly reduced the expression of Ccl2, Il6, and Tnf- (Tnf) (Figure 5B-D), confirming that AngII inducible Giver can indeed upregulate inflammatory genes. Furthermore, Giver knockdown inhibited AngII-induced Nox1 expression at both RNA (Figure 5E) and protein levels (Figure 5F). Conversely, exogenously (Figure 5G-J) and endogenously (by CRISPR-on) (Figure 5K-N) overexpressing Giver markedly enhanced expression of Ccl2, Il6, Tnf, and Nox1. Together, these results indicate that AngII inducible Giver enhances the expression of distally located target genes (Online Table VIII) that are closely associated with inflammation (Ccl2, Il6, and Tnf) and oxidative stress (Nox1).

Figure 5. Giver regulates pro-inflammatory and oxidative stress genes by altering chromatin states.

(A), Circos plot showing genes either upregulated (red) or downregulated (green) after Giver knockdown in RVSMC. Upregulated and downregulated genes are connected to the Giver locus on chr5 by blue lines. Outer circle represents chromosome ideograms and inner circles show histograms (red color) representing log2FC of each gene. Only the top 25 downregulated and upregulated genes are shown. (B-F), RT-qPCR validation of indicated genes (B-E) and Nox1 protein levels quantified by western blot (F) in siNTC- or siGiver-transfected RVSMC treated ± AngII. (G-J), RT-qPCR validation of indicated genes in RVSMC transfected with Giver expression vector (pGiver) versus empty vector (pEV). (K-N), RT-qPCR validation of indicated genes in RVSMC transfected with pAC154-dual-dCas9VP160-sg vector without sgRNA (EV), or vectors expressing sgRNAs targeting Giver promoter (PR) or exons (EX). (O-V), Bar graphs represent Pol II and H3K27me3 enrichments on the Ccl2 (O and S), Tnf (P and T), Nox1 (Q and U), and Rplpo (control) (R and V) promoters respectively in pEV and pGiver transfected RVSMC. Data represented as percent input. Mean+SEM; n=3 (for B-E and K-N); n=2 (for F); n= 6 (for G-J); n=3–4 (for O-V); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 using one-way ANOVA Tukey’s multiple comparison tests (for B-E) and Dunnett’s multiple comparison tests (K-N) and unpaired two-tailed t-tests (for G-J and O-V).

Next, to investigate whether epigenetic mechanisms are involved in Giver-mediated regulation of these genes, we performed ChIP assays with antibodies specific to RNA Polymerase II (Pol II) and repressive chromatin mark H3K27me3 after Giver overexpression in RVSMC. Giver overexpression increased Pol II enrichment but decreased H3K27me3 enrichment on Ccl2, Tnf, and Nox1 promoters (Figure 5O-Q and S-U). Rplpo promoter was used as negative control (Figure 5R and V).

Since Nox1 expression was enhanced, we next examined whether Giver can functionally promote oxidative stress. We measured intracellular reactive oxygen species (ROS) with fluorescent probes DHE and H₂DCFDA after transfection with siNTC or siGiver. Giver knockdown attenuated AngII-induced intracellular ROS production in RVSMC, as determined by decreased red fluorescence (Figure 6A-B). Furthermore, quantification of H₂DCFDA fluorescence intensity showed similar reduction after Giver knockdown (Figure 6C), thus confirming Giver can mediate AngII-induced ROS production. We also performed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and 5-bromo-2’-deoxyuridine (BrdU) incorporation assays to investigate the effects of Giver on cell proliferation. As shown in Figure 6D and 6E, Giver siRNA also attenuated AngII-induced proliferation in RVSMC. Together, these data demonstrate that Giver can mediate AngII-induced inflammatory gene expression, oxidative stress, and proliferation in VSMC.

Figure 6. Giver knockdown inhibits AngII-induced oxidant stress and cell proliferation.

(A), Representative images of DHE staining in RVSMC with indicated treatments. Hoechst and DHE shown in blue and red respectively. (B), Quantification of DHE fluorescent signals using Image J Pro software. (C), Mean fluorescence intensity of H₂DCFDA staining quantified by flow cytometry. (D-E), Cell proliferation of RVSMC was quantified by MTS (D) and BrdU incorporation (E) assays. Mean+SEM; n=4–6 (for A-B); n=3 (for C); n=12 (for D-E); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 using one-way ANOVA Tukey’s multiple comparison tests.

Giver interacts with the nuclear paraspeckles and splicing factor, non-POU domain-containing octamer-binding protein (NONO).

To evaluate potential mechanisms by which Giver RNA regulates key VSMC genes and functions, we performed RNA-pull down assays to identify Giver-interacting proteins. Giver sense and anti-sense RNAs were synthesized by in vitro transcription and then biotinylated. RNA integrity and biotinylation efficiency were checked by denaturing gel electrophoresis and dot blots (Online Figure IXA-B). Biotinylated sense and anti-sense probes were incubated with RVSMC nuclear extracts and RNA-bound complexes separated on an SDS-PAGE gel (Figure 7A). Protein bands from four different regions were subjected to mass-spectrometry analysis from sense and anti-sense lanes. Results revealed that multiple proteins are specifically associated with Giver sense strand but not anti-sense (Online Figure X). STRING database was used to identify the common protein complexes enriched among the Giver interacting proteins and their key functions (Figure 7B). Giver-interacting proteins were enriched in RNA binding proteins like NONO, and proteins involved in various RNA functions such as splicing and ribosome biogenesis. Interestingly, proteins in chromatin modification and remodeling complexes were also bound to Giver (Figure 7B). Specifically, RuvB Like AAA ATPase 1 (RUVBL1) and WD repeat-containing protein 82 (WDR82) are subunits of the INO80 chromatin remodeling complex and SET1 methyltransferase complex respectively (Online supplement Figure XIA). Moreover, histone-binding protein (RBBP7), AT-rich interactive domain-containing protein 4B (ARID4B) and chromodomain-helicase-DNA-binding protein 3(CHD3) are subunits of repressor complexes such as Sin3A-HDAC complex (Online supplement Figure XIB) suggesting Giver RNA can have positive and negative effects on gene expression. To validate some of these interactions, we performed RNA Immunoprecipitation (RIP) assays with NONO, WDR82, RBBP7 antibodies and control IgG. Results confirmed specific interaction of Giver with NONO, but not with other two interacting partners, possibly due to unavailability of rat-specific antibodies for these two proteins (Figure 7C, Online Figure XII). NONO did not co-precipitate with control Ppia. Interaction with NONO was also confirmed by RNA-pull-down followed by western blot (Figure 7D). Together, these data confirm NONO interacts with Giver and may mediate some molecular functions of Giver.

Figure 7. Giver interacts with nuclear proteins in RVSMC.

(A), Gel image showing proteins pulled down by biotinylated Giver sense and anti-sense RNA from RVSMC nuclear lysates. Box marked regions subjected to mass-spectrometry analysis. (B), Network generated using STRING database from Giver sense-strand-specific interacting proteins identified by mass-spectrometry. Human protein symbols for the indicated proteins were used to generate the network. Major enriched complexes are highlighted by circles. Solid lines indicate interactions within each complex. Thickness of the lines indicate confidence of interaction from literature. (C), NONO was immunoprecipitated from UV-crosslinked RVSMC nuclear lysates, and associated RNAs detected by RT-qPCR. (D), RNA pulldown followed by western blot with NONO antibody. (E-H), Bar graphs represent expression of indicated genes after siRNA-mediated depletion of NONO followed by AngII treatment in RVSMC. (I-K), Bar graphs represent H3K9ac, Pol II and H3K27me3 enrichment respectively on the Nr4a3 promoter in pEV and pGiver transfected RVSMC. Data represented as percent input. Mean+SEM; n=4 (for C and I-K); n=5–6 (for E-H); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 using one-way ANOVA Tukey’s multiple comparison tests (for C and E-H) and unpaired two-tailed t-tests (for I-K).

NONO is an RNA-binding protein belonging to the Drosophila behavior human splicing (DBHS) family of proteins including SFPQ and paraspeckle protein 1 (PSPC1). NONO regulates gene expression by multiple mechanisms.²⁸ NONO, together with SFPQ, PSPC1, hnRNPs, pre-mRNA splicing factors and lncRNA Neat1, forms paraspeckles²⁹ and is also involved in miRNA processing.³⁰ To evaluate the function of Giver-NONO complex, we examined effects of NONO knockdown in RVSMC (Figure 7E). NONO siRNA did not alter Giver expression under basal or AngII stimulated condition (Figure 7F), but, interestingly, it enhanced Nr4a3 expression under AngII induction and attenuated Il6 expression in both basal and AngII conditions (Figure 7G and H), showing that NONO knockdown phenocopies the effect of Giver knockdown on these genes (Figure 4F, 4K and 5C). Thus, Giver-NONO complex might be functionally involved in Giver-mediated gene regulation. Interestingly Giver overexpression decreased H3K9ac and Pol II enrichments on the Nr4a3 promoter (Figure 7I-J) H3K27me3 enrichment was unaltered (Figure 7K).

GIVER and NR4a3 transcripts are increased in arteries collected from patients with hypertension.

To investigate the clinical in vivo significance of GIVER in human vascular pathologies, we measured its levels in arteries from patients with hypertension versus controls, as well as hypertensive patients on ACE inhibitors (ACEI) or AngII receptor blocker (ARB) therapy (Online Table IX). Interestingly, GIVER expression was significantly increased in untreated hypertensive patients compared with non-hypertensive control subjects. In contrast, GIVER expression was markedly reduced in hypertensive patients treated with ACEIs or ARBs (Figure 8A). NR4A3 expression followed a similar pattern, with increases in untreated hypertensives and decreases in treated hypertensive subjects (Figure 8B). p47phox, a positive control for increased oxidant stress, especially in human hypertension³¹ also followed a similar pattern (Figure 8C). These data collectively implicate AngII induced GIVER and NR4A3 as important players in the pathogenesis of human hypertension likely through augmenting vascular oxidant stress, growth and inflammation.

Figure 8. Functional role of hGIVER in VSMC and in AngII-mediated hypertension. The Giver-Nr4a3 axis in VSMC dysfunction.

(A-C), GIVER, NR4A3 and p47phox are differentially expressed in arteries collected from hypertensive patients ± ACEI or ARB therapy. RNAs isolated from mammary artery trimmed residuals of patients undergoing CABG were subjected to RT-qPCRs to detect indicated genes. Mean with 95% CI; n=7 for non-hypertensive subjects, n=9 for hypertensive patients not on ACEI or ARB therapy, and n=4 for hypertensive patients on ACEIs or ARBs. (D-I), RT-qPCR validation of indicated genes in RVSMC transfected with human GIVER1 expression vector (pGIVER1) versus empty vector (pEV). Human GIVER1 transcript was characterized as described in Online Figure XIII. Mean±SEM; n=3 (for D-I); *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001 using one-way ANOVA Tukey’s (for A-B) and Dunn’s (for C) multiple comparison tests; and unpaired two-tailed t-tests (for D-I). (J), Schematic of the role of Giver in AngII-induced VSMC dysfunction. Activation of AT₁R by AngII induces NR4A3 in VSMC. NR4A3 induces GIVER via NBRE-element in its promoter. GIVER in turn inhibits NR4A3 indicating a negative feedback mechanism, but increases oxidative stress and inflammatory genes associated with VSMC dysfunction. Giver binding partners NONO as well as LRRFIP1, CHD3, RBBP7 and ARID4B might be involved in Giver-mediated repression of Nr4a3. On the other hand, GIVER increases NOX1 and inflammatory genes (likely via protein binding factors, Pol II recruitment, and histone modifications involved in chromatin remodeling and consequent chromatin relaxation) to promote oxidative stress, VSMC proliferation and inflammatory responses.

Putative hGIVER shows functional conservation with rGiver.

To assess whether human ortholog of GIVER can elicit similar phenotype as rGiver, we identified putative hGIVER transcripts (GIVER1-4) using RNA-seq data from HVSMC treated with AngII (unpublished) and publicly available HVSMC data¹⁶ aligned to the hg38 genome (See Online Methods and Figure XIIIA). We verified that AngII induces the expression of these putative hGIVER transcripts in HVSMC by RT-qPCR by using two different sets of primers (P1 and P2) (Online Figure XIIIB-C) similar to what we observed with the LiftOver identified hGIVER transcript. Interestingly, GIVER (P1) expression was also significantly increased in hypertensive patients and this was reduced in hypertensive patients treated with ACEIs or ARBs (Online Figure XIIID).

In order to further investigate the functional conservation between rGiver and hGIVER, we generated a putative hGIVER1 construct in pcDNA3.1 (pGIVER1) and overexpressed it in RVSMC, and examined candidate genes that we had found to be altered by rGiver in this system. Our data clearly showed human GIVER1 was overexpressed in RVSMC (Figure 8D) and also increased the expression of Ccl2 and Nox1 without affecting Tnf and Il6 (Figure 8E-H). Interestingly, ectopic GIVER1 overexpression in RVSMC did not alter Nr4a3 (Figure 8I). Together, these results suggest hGIVER shares some functional properties with rGiver.

DISCUSSION

In this study, we investigated the mechanisms of regulation and functional roles of a novel AngII-regulated rat lncRNA, Giver. We found that AngII induces the expression of Giver and its nearby protein-coding gene Nr4a3 in VSMC. AngII-induced Nr4a3 transactivated the expression of Giver through binding to the NBRE-related sequence 1515 bp upstream of Giver TSS. In turn, Giver transcript partially suppressed Nr4a3 expression but simultaneously up-regulated several target genes associated with inflammation and oxidative stress (trans actions), including Il6, Ccl2, Tnf, and Nox1, likely via interactions with key RNA binding proteins and chromatin remodeling complexes. In addition, Giver functionally promoted oxidative stress and cell proliferation in AngII-treated RVSMC. We further showed that GIVER was increased in AngII-treated HUVEC and HVSMC, but not in human THP-1 monocytes. Furthermore, GIVER and NR4A3 were markedly increased in arteries from human hypertensive patients.

LncRNAs are important epigenetic regulators that have recently been shown to be associated with vascular cell functions and vascular pathologies, acting via various mechanisms.^{8, 9, 12–16} In particular, nuclear lncRNAs can modulate the transcriptional activation or suppression of local (in cis) and/or distal (in trans) target genes through alterations in chromatin modifications including enhancers and super-enhancers at their loci and other mechanisms.^{13, 18, 19}

Here, we found that Giver is a 1.2 kb polyadenylated nuclear lncRNA located ~99 kb upstream of Nr4a3. Both Giver and Nr4a3 were upregulated in VSMC by various growth factors and pro-inflammatory cytokines in addition to AngII. Interestingly, Giver expression always peaked with or after that of Nr4a3, supporting a correlation between these neighboring genes. Their cross-talk regulation was also evident from the observation that rGiver promoter (−3026 to +1 bp) had several NBRE-related motifs. Further studies revealed that an NBRE-related sequence containing inverted repeat AAATACCA (−1527/−1515 bp)⁶ was involved in promoter transactivation, implying that Nr4a3 homodimers may transactivate Giver in AngII-treated RVSMC. These data also reveal a novel nuclear-receptor mediated regulation of a vascular lncRNA. On the other hand, treatment with siGiver enhanced Nr4a3 expression in AngII-treated RVSMC, whereas endogenous Giver overexpression (with CRISPR-on) inhibited Nr4a3. These data collectively suggest that Giver forms a negative regulatory loop with Nr4a3, with AngII induced Nr4a3 promoting Giver expression, and then Giver partially suppressing Nr4a3 expression in AngII-treated RVSMC (Figure 8J).

NR4a receptors function as constitutively active and ligand independent receptors and their transactivation activity is primarily regulated by extracellular stimuli that alter their expression and posttranslational modifications.^{5, 6} However, to our knowledge, there are no reports showing lncRNAs regulating the expression of NR4a receptors. Therefore, our observation that AngII inducible Giver partially suppressed Nr4a3 transcription reveals new lncRNA-dependent regulatory mechanisms of these genes.

In addition to the effects of Giver on its neighboring gene Nr4a3, we also explored its trans-effects on other target genes through unbiased microarray analysis. Compared with siNTC-transfected RVSMC, Nox1 was among the top ten differentially expressed genes in siGiver-transfected cells with or without AngII stimulation. ROS production in VSMC dysfunction and hypertension is well established, including the role of AngII as a major inducer of ROS in VSMC. Many of the pleiotropic vascular effects of AngII, such as inflammation and cell proliferation, are mediated through NAD(P)H oxidases (NOXs), including Nox1.³² Nox1 can mediate the hypertensive response to AngII,³³ again emphasizing the significance of the AngII-Nox1 axis. Our data suggests that Nox1 may be one of the key effectors of Giver-mediated signaling and vascular pathology. In support of this, IPA analysis showed significant enrichment of genes and pathways associated with oxidative stress, inflammation, and cell proliferation networks among those altered by siGiver. Importantly, we demonstrated the functional roles in VSMC pathology, because Giver knockdown attenuated AngII-induced ROS generation (likely due to Nox1 downregulation), and decreased cell proliferation in RVSMC. Thus, GIVER may promote cell proliferation and inflammation in VSMC through Nox1-mediated signaling pathways.

Examination of protein interacting partners of Giver RNA provided valuable clues about the potential mechanisms by which Giver promotes the expression of inflammatory genes and Nox1, while also suppressing Nr4a3. RNA-pull down coupled to mass-spectrometry revealed several proteins related to RNA function such as splicing, nuclear paraspeckles, ribosome biogenesis and translation as Giver interacting partners. We validated the interaction of the nuclear paraspeckle protein NONO with Giver. Interestingly, NONO knockdown had similar effects on Nr4a3 and Il6 gene expression as that of Giver knockdown, suggesting Giver-NONO complex may regulate these genes. NONO and SFPQ are known corepressors that interact with the well-known Sin3A-HDAC repressive complex.³⁴ Giver interacting partners also included RBBP7, CHD3 and ARID4B which are subunits of the Sin3A-HDAC complexe. Since we observed Giver negatively regulates Nr4a3 expression, after upregulation by AngII via Nr4a3, Giver might recruit these repressive complexes to the Nr4a3 promoter though interactions with NONO, SPFQ and other proteins, as part of an adaptive negative feedback loop (Figure 8J). Interestingly, leucine-rich repeat flightless-interacting protein1 (LRRFIP1), also identified as a Giver interacting protein, is an RNA binding protein that binds to GC rich sequences and functions as transcriptional repressor by binding to EZH2 in the PRC2 complex.^{35, 36} LRRFIP1 is induced by PDGF and TNF and can downregulate TNF, PDGF and epidermal growth factor receptor (EGFR) expression after initial burst of signaling.^{36, 37} As LRRFIP1 is also induced by AngII, similar mechanisms may downregulate Nr4a3 in VSMC after its initial surge (1h) of induction by AngII. Nr4a3 promoter harbors GC rich sequences that could be bound by LRRFIP1, Giver and repressive complexes to repress Nr4a3 expression. Ectopic Giver overexpression increased Giver transcript levels to a much greater magnitude (almost 60 fold) compared to its endogenous overexpression by CRISPR-on or AngII treatment (2–3 fold) (Figure 4G versus 4J and 1B). This high expression of Giver might facilitate the binding of such repressive complexes to Giver RNA and subsequent recruitment to the Nr4a3 locus, resulting in decreased H3K9ac and Pol II enrichment (Figure 7I-J), but not sufficient enough to significantly decrease Nr4a3 expression (Figure 4H).

We also found key proteins related to active chromatin complexes such as SET1 (Online Figure XIA) among Giver interacting proteins. LncRNAs can regulate transcription by recruiting regulatory protein complexes in trans by RNA-protein interactions.³⁸ Such interactions are possibly involved in Giver mediated increases Nox1 and inflammatory gene expression via chromatin remodeling and epigenetic mechanisms (Figure 8J). This is indirectly supported by ChIP data showing increased Pol II and decreased H3K27me3 enrichment at these genes in Giver overexpressing cells (Figure 5O-V). Therefore, Giver might have negative effects on Nr4a3 via cis action, but positive effects on the expression of other genes in trans, through interactions with key proteins (like NONO) and other mechanisms including histone modifications. Additional experiments are needed to test these mechanisms systematically. Of note, we recently showed lncRNA Dnm3os has trans-actions on inflammatory genes in macrophages through RNA-protein interactions with nucleolin.³⁹

ACEIs and ARBs, among the first-line of anti-hypertensive drugs, directly alter the production and functions of AngII.⁴⁰ However the epigenetic mechanisms of action of ACEI/ARBs remain largely unknown. We found correlations between lncRNA GIVER levels and ACEI/ARB therapy. Expressions of GIVER, NR4A3 and p47phox (positive control for oxidant stress in human hypertension³¹) were increased in mammary arteries collected from hypertensive patients relative to controls, but were restored to normal levels in hypertensive patients on ACEIs or ARBs. To our knowledge, this is the first study showing correlation between lncRNAs and antihypertensive therapy. In addition, hGIVER was induced by AngII in HVSMC and HUVEC, but not in THP-1 monocytes, indicating specific regulation in vascular cells. Importantly, alterations in GIVER transcript was always accompanied by prior or simultaneous changes of NR4A3 RNA, implying that GIVER may also be regulated by NR4A3 in human vasculature. Notably, we used a combination of in silico transcriptome data analyses coupled with experimental approaches, including cloning and functional assays, to identify putative hGIVER transcripts showing functional conservation, at least in part, with rGiver (Online Figure XIII and Figure 8D-I).

In summary, we report several new findings, including the mechanisms of regulation of a novel AngII-induced lncRNA GIVER by a nuclear receptor, its functional roles in promoting inflammatory gene expression (via epigenetic mechanisms), oxidative stress and proliferation, identification of Giver interacting proteins and association of the GIVER-NR4A3 pathway with hypertension as well as RAS-associated antihypertensive therapy. The in-depth molecular mechanisms by which Giver interacting proteins modulate its functions needs further investigation. Notwithstanding, the current observations enhance our understanding of the complex interplay between lncRNAs and nuclear receptors such as NR4A3 in the pathogenesis of hypertension and CVDs, which, in turn, could lead to new therapeutic interventions for hypertension.

What Is Known?

• Angiotensin II (AngII), a key player in hypertension and other cardiovascular diseases, promotes growth and inflammatory responses in vascular smooth muscle cells (VSMC).

• AngII exerts its pathological effects in VSMC via the AngII type 1 receptor and several downstream signaling pathways, transcription factors, and protein-coding genes.

• AngII also regulates microRNAs, long noncoding RNAs (lncRNAs), enhancers and super-enhancers in VSMC. These epigenetic regulators contribute to inflammatory and growth promoting effects of AngII.

What New Information Does This Article Contribute?

• AngII induces a novel lncRNA Giver in VSMC which negatively regulates its nearby protein coding gene Nr4a3 by interacting with non-pou domain-containing octamer-binding protein (NONO).

• Giver also regulates inflammation, oxidative stress and proliferation in VSMC by altering the expression of key genes involved in these processes via epigenetic trans mechanisms.

• Human GIVER expression was increased in vivo in hypertensive patients and attenuated in hypertensive patients treated with Angiotensin Converting Enzymes Inhibitors or Angiotensin Receptor Blockers. Rat Giver and human GIVER showed functional similarities.

LncRNAs are emerging as novel epigenetic regulators of gene expression and disease states. Although recent studies show that AngII induces the expression of several lncRNAs in VSMC, their functions and mechanisms of action are unclear. Here, we characterized a novel AngII regulated lncRNA Giver in VSMC and examined the molecular mechanisms by which it promotes VSMC dysfunction. We found that Giver and its nearby nuclear receptor gene Nr4a3 are coordinately altered by AngII both in vitro and ex vivo, and Giver is transcriptionally regulated by Nr4a3. Using gain- and loss-of-function approaches, we discovered that lncRNA Giver suppresses the expression of Nr4a3 via negative feedback mechanisms, but increases the expression of oxidative and inflammatory genes involved in VSMC dysfunction via chromatin remodeling and epigenetic mechanisms. We found that Giver interacts with nuclear paraspeckles, splicing factor NONO and other corepressors to inhibit Nr4a3 expression. Notably, we demonstrate that rat Giver shares functional conservation with the human GIVER ortholog in regulating VSMC dysfunction. Furthermore, we found GIVER levels are enhanced in arteries of hypertensive subjects, and attenuated in hypertensive patients treated with angiotensin receptor blockers. These results illustrate new lncRNA-GIVER dependent molecular mechanisms of AngII action that could serve as novel targets for anti-hypertensive therapy.

Nonstandard Abbreviations and Acronyms:

ACEI

angiotensin converting enzyme inhibitor

AngII

angiotensin II

ARB

angiotensin receptor blocker

AT₁R

AngII type-1 receptor

BP

blood pressure

BrdU

5-bromo-2’-deoxyuridine

CABG

coronary artery bypass grafting

CVD

cardiovascular diseases

DHE

dihydroethidium

FBS

fetal bovine serum

H₂DCFDA

2’,7’-dichlorodihydrofluorescein diacetate

HG

high glucose

IPA

Ingenuity Pathway Analysis

lncRNA

long non-coding RNA

miRNA

microRNA

MTS

3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt

NBRE

nerve growth factor-induced protein B-responsive element

ncRNA

non-coding RNA

PDGF-BB

platelet-derived growth factor-BB

RAAS

renin-angiotensin-aldosterone system

ROS

reactive oxygen species

VSMC

vascular smooth muscle cell

RIP

RNA immunoprecipitation

NONO

non-POU domain-containing octamer-binding protein

Pol II

RNA Polymerase II

H3K27me3

histone H3 trimethylation on lysine 27