Cardiovascular diseases (CVDs), such as hypertension and atherosclerosis, are leading causes of morbidity and mortality worldwide ^[1]. Vascular smooth muscle cells (VSMCs) in the arterial wall maintain vascular tone and blood pressure and are under the control of the renin–angiotensin system (RAS)-Angiotensin II (AngII) system.

In CVD or vascular injury, dysregulated growth factor and AngII signaling promotes VSMCs to switch from a contractile to synthetic phenotype [3]^[2]. The synthetic phenotype manifests in increased VSMC proliferation, hypertrophy, migration, inflammation and the key processes associated with the pathogenesis of arterial stenosis/restenosis, hypertension and atherosclerosis [4,5,6,7]^[3][4][5][6]. Furthermore, the synthetic VSMCs tend to transform into chondrocyte-like cells, which promotes extracellular calcium deposition and vascular dysfunction associated with these pathologies [8,9,10]^[7][8][9]. Aggrecan (Acan) is an extracellular matrix protein that is prominent in chondrocytes during cartilage formation and is upregulated in aortic VSMCs after injury [10]^[9]. The transcription factor (TF) Sox9, which regulates chondrogenesis, is associated with VSMC synthetic/chondrocyte phenotype and promotes extra-cellular matrix (ECM) alterations and calcium deposition [11]^[10]. However, the mechanisms involved in AngII-mediated phenotypic transformation of VSMC to chondrocyte-like cells are not well understood.

Long non-coding RNAs (lncRNAs) are a group of non-coding RNAs (ncRNAs) that are more than 200 nucleotides in size and are processed like protein coding mRNAs but lack protein-coding potential [12]^[11]. LncRNAs have diverse functions and regulate gene expression at the level of transcription through the interaction with and recruitment of TFs, chromatin modifier proteins and ribonucleoproteins to specific target gene loci, or via the post-transcriptional regulation of microRNAs and signaling proteins [13]^[12]. Genome-wide association studies (GWAS) identified several single nucleotide polymorphisms (SNPs) associated with CVDs that reside in the lncRNA loci [14]^[13]. LncRNAs regulate various physiological and pathological processes [15]^[14]. In VSMCs they regulate cell proliferation, migration, reactive oxygen species (ROS) production and inflammation, key factors associated with CVDs [16,17]^[15][16]. WResearchers identified the first lncRNAs regulated by AngII in rat VSMCs (RVSMCs) using integrated analysis of RNA-seq data with ChIP-seq datasets from histone H3K4me3 and H3K36me3 profiling [18]^[17]. Since then, several VSMC lncRNAs such as SENCR, MYOSLID and SMILR were described and found to play key roles in CVDs [19,20,21]^[18][19][20].

2. Alivec Is an AngII-Induced lncRNA Adjacent to Chondrogenic Gene Acan in RVSMCs

ThisHere study analyzed RNA-seq data previously generated in oura laboratory from RVSMCs treated with AngII (100 nM, 3 h) [18]^[17] using STAR aligner and observed that a previously identified novel lncRNA (lnc Ang26), which named Alivec, was highly induced by AngII (Figure 1A). To further characterize the Alivec locus, wresearchers integrated the RNA-seq data with histone H3K27ac (enhancer mark) ChIP-seq data from AngII treated RVSMCs [24]^[21]. Combined RNA-seq and ChIP-seq data showed that the lncRNA Alivec locus overlaps with an AngII-induced H3K27ac enriched region (Figure 1B). Alivec has 3 exons and the gene is located on rat chromosome 1 adjacent (117 kb distance) to the protein-coding gene Acan (Figure 1B). RNA-seq analyses also showed that the expression of the nearby gene Acan, was likewise increased by AngII. Furthermore, RT-qPCR validation showed that RVSMCs exposed to AngII displayed marked induction of Alivec expression (up to 30-fold) within 3 h of treatment; this persisted even at 6 h compared to the control cells (Figure 1C). Under the same conditions, the induction of Acan was also observed (Figure 1D), suggesting a potential role for Alivec in the regulation of Acan expression by AngII. This was interesting, as Acan codes for the protein aggrecan, which is known to be induced by growth factors and cytokines and is also a key biomarker of chondrogenesis associated with VSMC dysfunction in CVDs [31]^[22]. Next, researchers performed experiments to further characterize Alivec. Rapid amplification of cDNA end (RACE)-PCR experiments verified the 5′ and 3′ ends of Alivec and defined the total transcript size to be 2275 nucleotides (Supplementary Figure S1A,B and Supplementary Table S2). Considering the localization of lncRNAs in the nucleus or cytoplasm can determine their functions, [32]^[23] wresearchers examined the cellular localization of lncRNA Alivec. In AngII-treated RVSMCs, sub-cellular fractionation followed by RT-qPCR showed that Alivec is distributed in the nucleus and cytosol (Figure 1E). Ppia and a lncRNA Neat1 served as controls for cytoplasmic and nuclear fractions, respectively (Figure 1E). RNA–FISH experiments with branched DNA probes, further confirming nuclear and cytoplasmic localization of Alivec, as indicated by the presence of distinct spots/foci distributed in both compartments (Figure 1F). These spots were not visible in the absence of the probes (Supplementary Figure S1C). The protein-coding potential analysis of Alivec (coding potential calculator version 2.0, CPC2) showed that it had a coding probability of 0.31, classifying it as a non-coding transcript. The lack of coding potential was confirmed by in vitro transcription/translation assays using pcDNA Alivec plasmids, which showed no detectable peptide product from Alivec, as compared to the positive luciferase control (Supplementary Figure S1D,E). Together, these results indicate that Alivec is an AngII-induced lncRNA in RVSMCs.

3. AngII-induces Alivec and Acan Expression via Activation of AT1R and Src Kinase

AngII, through the activation of AT1R, induces multiple signaling pathways to regulate downstream gene expression and alter VSMC function [2]^[24]. To elucidate the role of AT1R signaling in regulating Alivec and Acan expression, wresearchers pre-treated RVSMCs with the AT1R antagonist, losartan (10 µM, 30 min), followed by AngII (100 nM). Losartan abrogated the AngII-mediated induction of Alivec (Figure 2A). Acan expression was also attenuated by losartan (Figure 2B). Pre-treatment of RVSMCs for 30 min with specific inhibitors of key signaling kinases, ERK1/2 (U0126, 10 µM) and Src (PP1,10 µM), significantly reduced AngII-induced Alivec expression. Conversely, inhibitors of the p38 MAP kinase (SB202190, 5 µM) and JAK (Inhibitor I, 10 µM), had minimal effect on Alivec expression (Figure 2C). Interestingly, the AngII-mediated induction of Acan was significantly suppressed by inhibition of the Src kinase, and to a lesser extent, by inhibitors of ERK1/2 and JAK (Figure 2D). These results demonstrate key roles of AT1R signaling and downstream kinases in the regulation of Alivec and Acan in RVSMCs. It also was determined if Alivec is induced by other VSMC growth factors, such as PDGF, and the inflammatory cytokine TNF-α. RVSMCs treated with PDGF (10 ng/mL) and TNF-α (10 ng/ml, 3 and 6 h) showed increased Alivec and Acan mRNA levels, although with varying kinetics (Figure 2E–H) suggesting a general effect. Together, these results show that Alivec and Acan are upregulated by not only AngII, but by other growth factors and inflammatory cytokines that might co-operate to augment VSMC dysfunction upon vascular injury.

4. Alivec Knockdown Attenuates AngII-Induced Upregulation of Genes Associated with Chondrogenesis

AngII regulates the expression of inflammatory, fibrotic and calcification-related genes in VSMC [2]^[24]. To further characterize the role of Alivec in the regulation of AngII-induced gene expression in RVSMCs, a locked nucleic acid (LNA)-modified anti-sense oligonucleotide (ASO) GapmeRs-mediated knockdown of Alivec was performed. Three GapmeRs targeting Alivec were tested to assess their efficacy in RVSMCs (Supplementary Figure S2A). Results showed that GapmeR3 (denoted as AlivecGap) achieved maximum reduction (~60%) in AngII-induced Alivec expression, as compared to the control GapmeR (NCGap) (Figure 3A and Supplementary Figure S2B). RVSMCs were transfected with AlivecGap or NCGap and treated with or without AngII. RNA extracted from these cells was subjected to microarray expression profiling (Supplementary Figure S3A,B). After Alivec knockdown, researchers identified 1169 differentially expressed genes in untreated RVSMCs (676 downregulated and 493 upregulated), and 1294 differentially expressed genes in AngII-treated RVSMCs (664 downregulated and 630 upregulated), which included several chondrogenic genes (Figure 3B). Gene ontology (GO) analysis of downregulated genes showed enrichment of biological processes, such as cell adhesion and the circulatory system (Figure 3C), which are important functions of VSMC and the cardiovascular system. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed enrichment of pathways involved in mucin type O-glycan biosynthesis, nitric oxide second messenger cGMP signaling and vascular smooth muscle contraction (Figure 3D) that could be associated with VSMC functions and hypertension. RT-qPCR validation of microarray data confirmed downregulation of Acan and several other chondrogenic genes, including Tnfaip6, Runx1, Olr1 and Spp1 (Figure 3E–I), after Alivec knockdown in RVSMCs. In addition, Acan downregulation is consistent with the known role of lncRNAs in regulating adjacent genes (Figure 3B). Conversely, in gain-of-function experiments, transient overexpression of Alivec increased mRNA levels of Acan, Runx1, Tnfaip6, Olr1 and Runx2, relative to the controls (Figure 4A–F). Together, these results demonstrate that lncRNA Alivec plays a key role in the regulation of AngII-induced chondrogenic genes in RVSMCs.

5. Alivec Mediates a Chondrogenic/Osteogenic Phenotype in RVSMCs

Alcian blue stains glycosaminoglycan proteins, including aggrecan, that are associated with the ECM and chondrogenic differentiation [33]^[25]. Relative to control, AngII-treated RVSMCs showed increased alcian blue staining, and this was significantly decreased by Alivec knockdown with GapmeR (Figure 4G). Conversely, overexpression of Alivec increased alcian blue staining (Figure 4H). These data demonstrate that Alivec regulates expression of several AngII-induced chondrogenic genes, including nearby Acan, and promotes a chondrocyte phenotype in RVSMCs.

6. Transcription Factor Sox9 Controls Alivec Expression in RVSMCs

Transcription factor (TF) motif analyses of 500 bases upstream of the Alivec transcription start site (TSS) showed enrichment of ten TFs, including Sox9 (Figure 5A). Sox9 regulates chondrogenesis and osteogenesis in mesenchymal stem cells [34]^[26]. Wresearchers examined Sox9 interaction with the Alivec promoter in RVSMCs transfected with a Sox9 expression plasmid (pcDNASox9) and a control vector (pcDNACtrl), using chromatin immunoprecipitation (ChIP) assays with the Sox9 antibody. ChIP-qPCR showed enrichment of Sox9 in the predicted Sox9-binding region, upstream of the Alivec TSS, as compared with the control pcDNACtrl plasmid-transfected cells (Figure 5B). Transfection of RVSMCs with the siRNAs targeting Sox9 (siSox9), reduced the Sox9 protein and transcript levels in control- and AngII-treated cells (Figure 5C,D). Sox9 knockdown also decreased the AngII-induced expression of Alivec and Acan (Figure 5E,F). Conversely, the overexpression of Sox9 using the pcDNASox9 plasmid in RVSMCs increased Alivec and Acan vs. the control vector-transfected cells (Figure 5G–I). These results demonstrate that Sox9 can regulate Alivec and Acan expression in response to AngII in RVSMCs.

7. Alivec RNA Interacts with hnRNPA2B1 as well as with Tropomyosin alpha-3 Chain, a Protein with Putative Association with the Contractile Phenotype of RVSMCs

LncRNAs can regulate transcription, gene expression and cellular phenotype through interactions with proteins [35,36]^[27][28]. WResearchers performed RNA-pulldown assays with Alivec, followed by mass spectrometry, and found a number of proteins associated with Alivec, relative to negative control. STRING analysis demonstrated that the Alivec interacting proteins were associated with VSMC contractile functions, nuclear membrane organization and regulation of gene expression (Figure 6A). One of these proteins, a tropomyosin alpha-3 chain (Tpm3) [37]^[29] was noteworthy, due to the known roles of alpha-tropomyosin isoforms in VSMC contractile functions and gene regulation [38,39]^[30][31]. RNA–protein interaction prediction (RPISeq) software showed that the Alivec-Tpm3 RNA–protein interaction had a positive interaction probability of 0.75 (>0.5 considered positive). WResearchers then performed RNA-pulldown, followed by Western blot analysis, in order to validate the Tpm3 association with Alivec (Figure 6B), which confirmed ourthe mass spectrometry results. Specific interaction of Alivec with Tpm3 was also supported by RNA-immunoprecipitation (RNA-IP), using an antibody against Tpm3. No interaction was seen with Gapdh mRNA and H19 lncRNA (negative controls, Figure 6C). In addition, mass spectrometry showed that Alivec interacts with the RNA-binding protein, heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1), which was validated by RNA-pulldown, followed by Western blotting (Figure 6B, lower panel). These results indicate that lncRNA Alivec mediates AngII-induced VSMC dysfunction via interaction with both cytoplasmic and nuclear proteins.

8. AngII Treatment Increases Aortic Expression of Alivec in Rats

Next, study examined whether AngII upregulates the expression of Alivec and Acan in vivo in rats. Male Sprague–Dawley rats (12-weeks-old), infused with AngII (200 ng/kg/min, four weeks), showed the expected increase in systolic blood pressure (SBP) compared to control vehicle (PBS) infused rats (Figure 7A). Aortic thickening was noted in AngII-infused animals relative to the controls (Figure 7B). Immunohistochemical staining showed marked increases in aggrecan and Runx1 proteins and decreases in the smooth muscle contractile proteins α-SMA and SM22 alpha (Figure 7B). Furthermore, mRNA levels of Alivec, Acan and Runx1 were significantly increased in vessels from the AngII group (Figure 7C–E).