Angiotensin II-Induced Long Non-Coding RNA Alivec Regulates Chondrogenesis in Vascular Smooth Muscle Cells

Long non-coding RNAs (lncRNAs) play key roles in Angiotensin II (AngII) signaling but their role in chondrogenic transformation of vascular smooth muscle cells (VSMCs) is unknown. We describe a novel AngII-induced lncRNA Alivec (Angiotensin II-induced lncRNA in VSMCs eliciting chondrogenic phenotype) implicated in VSMC chondrogenesis. In rat VSMCs, Alivec and the nearby gene Acan, a chondrogenic marker, were induced by growth factors AngII and PDGF and the inflammatory cytokine TNF-α. AngII co-regulated Alivec and Acan through the activation of AngII type1 receptor signaling and Sox9, a master transcriptional regulator of chondrogenesis. Alivec knockdown with GapmeR antisense-oligonucleotides attenuated the expression of AngII-induced chondrogenic marker genes, including Acan, and inhibited the chondrogenic phenotype of VSMCs. Conversely, Alivec overexpression upregulated these genes and promoted chondrogenic transformation. RNA-pulldown coupled to mass-spectrometry identified Tropomyosin-3-alpha and hnRNPA2B1 proteins as Alivec-binding proteins in VSMCs. Furthermore, male rats with AngII-driven hypertension showed increased aortic expression of Alivec and Acan. A putative human ortholog ALIVEC, was induced by AngII in human VSMCs, and this locus was found to harbor the quantitative trait loci affecting blood pressure. Together, these findings suggest that AngII-regulated lncRNA Alivec functions, at least in part, to mediate the AngII-induced chondrogenic transformation of VSMCs implicated in vascular dysfunction and hypertension.


Introduction
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. AngII, the primary effector of the RAS pathway, is a potent vasoconstrictor and regulator of blood pressure. Dysregulation of RAS or abnormal AngII signaling is implicated in hypertension and atherosclerosis [2].

RNA Isolation and RT-qPCR
Total RNA was isolated from the rat aortas, RVSMCs and HVSMCs, using TRIzol and an RNeasy mini kit (Qiagen, Germantown, MD, USA). cDNA was synthesized using a highcapacity cDNA reverse transcription kit (Thermo Fisher Scientific, Carlsbad, CA, USA). RT-qPCR was performed using SYBR green master mix (Applied Biosystems, Foster City, CA, USA) and analyzed on a 7500 Fast Real Time PCR system (Thermo Fisher Scientific). Relative gene expression was analyzed using the 2 −∆∆ Ct method and normalized to Ppia (rat) and GAPDH (human), as described [23,24]. Sequences of primers used are listed in Supplementary Table S1.

Cellular Fractionation
Cytoplasmic and nuclear fractions from RVSMCs were purified and RNA isolated using a cellular fractionation kit (Norgen, ON, Canada). Briefly, RVSMCs were incubated on ice with cold lysis solution and centrifuged to separate cytoplasmic and nuclear components [23]. RNA was isolated from each fraction followed by RT-qPCR, as described above.

RNA Fluorescence In Situ Hybridization (RNA-FISH)
RNA-FISH was performed to determine the subcellular localization of Alivec using a ViewRNA ISH Cell Assay Kit (Affymetrix, Santa Clara, CA, USA), as described [23]. Branched DNA signal amplification probes manufactured by Affymetrix eBiosciences (Thermo Fisher Scientific) were used to target Alivec. RVSMCs were plated in 4-chamber slides (LAB-TEK Nunc, Rochester, NY, USA) in M199 complete medium, treated with AngII (100 nM) for 3 h and fixed with 4% formaldehyde, with probes and signal amplification reagents added. Cells stained with Ppia probes served as a positive control and cells without a probe served as a negative control. Images were captured with a Zeiss Observer Z1 wide-field microscope and processed using ZEN Blue software (Zeiss, Oberkochen, Germany) with settings kept constant across all the samples.

5 and 3 Rapid Amplification of cDNA Ends (RACE)
To define the specific ends of Alivec transcript, 5 and 3 RACE-PCRs were performed using a FirstChoice RLM-RACE kit (Thermo Fisher Scientific). The PCR products were Sanger sequenced and, using SnapGene software (GSL Biotech, Chicago, IL, USA), the sequences aligned to the Alivec genomic locus to define the ends. The coding potential of the full length Alivec sequence was determined using the coding potential calculator tool web-based portal version 2.0 (CPC2) [26].

In Vitro Transcription and Translation
The complete Alivec cDNA sequence (Supplementary Table S2) was synthesized and cloned into pcDNA3.1+ vector (Thermo Fisher Scientific) by a commercial vendor (Vector Builder Inc, Chicago, IL, USA) to generate a pcDNA3.1-Alivec construct. Then, the linearized pcDNA3.1-Alivec construct was subjected to in vitro transcription and translation assays to verify the coding potential of Alivec using the T7 TNT quick coupled transcription/translation system (Promega, Madison, WI, USA). Control pcDNA3.1 plasmid with luciferase expressing from the T7 promoter was used as a positive control and no plasmid template (Thermo Fisher Scientific) was used as a negative control. The translation products from these reactions were loaded on to SDS-PAGE gel and then transferred onto a positively charged nylon membrane. Protein products were detected with a streptavidin antibody and western blue reagent (Promega).

Transient Transfection of RVSMCs with Plasmids, GapmeRs and siRNAs
RVSMCs were transiently transfected with antisense-locked nucleic acid (LNA)modified GapmeRs (100 nM), targeting Alivec (AlivecGap) or a non-targeting control (NC-Gap) obtained from Qiagen, and small interfering RNAs (siRNAs, 10 nM) targeting Sox9 or the control non-targeting siRNAs (Horizon, Lafayette, CO, USA) using Lipofectamine RNAiMax (Thermo Fisher Scientific), as described [23]. Transfected cells were serum depleted for 24 h prior to the AngII treatment (100 nM, 3 h) and RNA was collected 48-72 h after transfection. RVSMCs were transiently transfected with expression plasmids for Alivec and pcDNA-Sox9 (a kind gift from Maike Sander, UCSD, San Diego, CA, USA) using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Sequences of siRNAs and GapmeRs used in this study are listed in Supplementary Table S3.

Affymetrix Gene Array Analyses
Microarray hybridization, data acquisition and the initial analysis were performed by the Integrative Genomics Core of City of Hope. Biotinylated cDNA derived from total RNA was hybridized with the Clariom S Assay GeneChip array for rat transcriptome wide gene expression profiling (Thermo Fisher Scientific). Three independent replicates were performed for each group of samples. Raw intensity data in CEL file format were imported into the Partek Genomics Suite (version 6.6, Partek Inc., St. Louis, MO, USA) and preprocessed and normalized using the Robust Multichip Average method. The probe sets with no or low expression (normalized log2 signal intensity less than 6) were removed from further analysis. Comparisons between NCGap and AlivecGap transfected at the basal level and AngII-treated RVSMCs were performed using the analysis of variance method in Partek. Statistically significant differentially expressed genes (DEG) were defined using the criteria of absolute fold change ≥1.20 and a p-value of <0.05. Biological functions and network analysis of DEG was carried out with TOPPGENE, DAVID and KEGG pathway tools. Transcription Factor Affinity Prediction (TRAP) web tools were used for analyzing the TF-binding motifs.

Identification of Rat lncRNA Alivec
Publicly available RNA-seq data (GSE38056) and ChIP-seq data (GSE95067), previously published by our laboratory [18,24], were used to identify lncRNA Alivec and the enrichment of H3K27ac overlapping Alivec locus in rat VSMCs. The RNAseq data from rat VSMCs treated ± AngII for 3 h were aligned to rat genome assembly rn4 (Baylor 3.4/rn4) with spliced transcript alignment to a reference (STAR, version 2.6.0.a) aligner tool using default parameters. Integrative Genomics Viewer was used to visualize the RNA-seq and ChIP-seq datasets.

Alcian Blue Staining to Determine Chondrogenic Phenotype
Following knockdown and the overexpression of Alivec, RVSMCs were incubated overnight with 0.1% alcian blue (Sigma-Aldrich, Burlington, MA, USA) in 0.1 M HCl. Cells were washed, bound and stain extracted with 6 M guanidinium hydrochloride for 8 h, with the absorbance read at 620 nm [27].

AngII-Infused Rat Model of Hypertension and Vasculopathy
Osmotic minipumps (Alzet model 2002, Cupertino, CA, USA) filled with AngII or vehicles were implanted subcutaneously in 12-week-old male Sprague-Dawley rats (3 rats/group). AngII was delivered at a rate of 200 ng/kg/min for 28 days [28]. During the final week of the experiment, blood pressure was measured using a tail cuff system (Visitech, Apex, NC, USA). At the end of the experiment, rats were humanely euthanized by CO 2 and aortas harvested for RNA isolation and immunohistochemistry.

Alivec RNA-Pulldown and Mass Spectrometry
Alivec RNA-pulldown assays were performed with lysates from RVSMCs treated with AngII, using published methods with some modifications [29]. The Alivec-expressing plasmid, pcDNA-Alivec, was used as a template in an in vitro transcription kit (Roche) to generate Alivec RNA. Alivec RNA or polyA RNA (the negative control) were biotin-labeled using an RNA 3 Desthiobiotinylation Kit (Thermo Fisher Scientific). RNA-pulldown assays were performed using the Pierce Magnetic RNA-protein pulldown kit (Thermo Fisher Scientific) following the manufacturer's protocol. Briefly, protein lysates (100 µg) from RVSMCs, treated with AngII (100 ng/mL, 3 h), were incubated with biotin-labeled Alivec or polyA RNA probes (100 pmol) and yeast tRNA (30 µg) at 4 • C for 2 h. The bound RNA-protein complexes were incubated with streptavidin beads for 2 more hours. The complexes were washed 5 times to remove non-specific binding proteins. Proteins were eluted using TRIS buffer and subjected to mass spectrometry (MS) analysis at the City of Hope Proteomics Core. The scaffold tool (Proteome Software Inc, Portland, OR, USA) was used to identify and validate the MS/MS-based peptides. Protein identifications were accepted if they contained at least 2 identified peptides and could be established with a minimum of 99.0% probability with the Scaffold local FDR algorithm. For validation of mass spectrometry results, eluted proteins were analyzed by Western blotting with antibodies against hnRNPA2B1 (1:1000) (Origene, Rockville, MD, USA) and Tpm3 (1:1000) (Genetex, Irvine, CA, USA) (ST III).

UV-RNA Immunoprecipitation (RIP) Assay
The assay was performed as described [30]. Briefly, 1.0 × 10 7 RVSMCs treated with AngII for 3 h were cross-linked with UV light using Stratalinker (1200 µjoules/cm 2 ) and lysed with lysis buffer. The lysates were diluted in RIP buffer and incubated with 5 µg each of anti-Tpm3 (Genetex) or rabbit IgG as the controls. The antibody-bound RNA-protein complexes were captured on magnetic protein G beads and bound RNA was isolated, followed by an RT-qPCR analysis.

Data Deposition
Affymetrix data are deposited in the Gene Expression Omnibus (accession number: GSE183857).

Statistical Analysis
All experiments were performed at least 3 times unless otherwise mentioned in the figure legend. Data were analyzed using GraphPad PRISM 8 (GraphPad, San Diego, CA, USA). The data were represented as the mean ± standard deviation (SD). A p-value <0.05 was considered statistically significant based on unpaired two-tailed t-tests for two groups and one-way ANOVA with Dunnett's or Tukey's multiple comparison tests for multiple groups. Normal data distributions were confirmed using the Shapiro-Wilk normality test.

Alivec Is an AngII-Induced lncRNA Adjacent to Chondrogenic Gene Acan in RVSMCs
We analyzed RNA-seq data previously generated in our laboratory from RVSMCs treated with AngII (100 nM, 3 h) [18] using STAR aligner and observed that a previously identified novel lncRNA (lnc Ang26), which we named Alivec, was highly induced by AngII ( Figure 1A). To further characterize the Alivec locus, we integrated the RNA-seq data with histone H3K27ac (enhancer mark) ChIP-seq data from AngII treated RVSMCs [24]. 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].
Next, we 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] we 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.

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]. To elucidate the role of AT1R signaling in regulating Alivec and Acan expression, we 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.
We also 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.

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]. 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, we 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.  cluded 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.

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]. 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.

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]. We examined Sox9 interaction with the Alivec promoter in RVSMCs transfected with a Sox9 expression plasmid (pcDNASox9) and a control vector (pcDNACtrl), using chromatin immunopre-cipitation (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 controland 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 vectortransfected cells (Figure 5G-I). These results demonstrate that Sox9 can regulate Alivec and Acan expression in response to AngII in RVSMCs.
as determined by RT-qPCR. Data presented as mean ± SD, one-way ANOVA followed by Tukey's post-hoc test and **** p < 0.0001 vs. indicated groups. (B) Volcano plot showing differentially expressed genes (orange color) in AngII-treated RVSMCs transfected with AlivecGap vs. NCGap. Labeled dots indicate genes involved in chondrogenesis. (C) Gene ontology (GO) analysis by the TOPPGENE tool of differentially-expressed (DE) genes showing the top 10 biological processes enriched in downregulated genes after Alivec knockdown. (D) KEGG pathway analysis of differentially-expressed (DE) genes, showing the top 10 molecular pathways affected in downregulated genes after Alivec knockdown. (E-I) RT-qPCR validation of indicated chondrogenic genes after Alivec knockdown in RVSMCs treated ± AngII (100 nM, 3 h). Data presented as mean ± SD, one-way ANOVA followed by Tukey's post-hoc test and ** p < 0.01 and *** p < 0.001 vs. indicated groups) n = 3 biological replicates.
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.   RT-qPCR analysis of indicated genes in RVSMCs, transfected with Sox9 expression plasmid (pcDNASox9) and pcDNACtrl control plasmid. Data represented as mean ± SD, n = 3 biological replicates, unpaired Student's t-test and * p < 0.05, *** p < 0.001 vs. control plasmid.

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]. We 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] was noteworthy, due to the known roles of alpha-tropomyosin isoforms in VSMC contractile functions and gene regulation [38,39]. 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). We then performed RNApulldown, followed by Western blot analysis, in order to validate the Tpm3 association with Alivec ( Figure 6B), which confirmed our 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.

AngII Treatment Increases Aortic Expression of Alivec in Rats
Next, we 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).

The Human ALIVEC Locus Contains ACAN Regulatory Elements and a Blood Pressure Quantitative Trait Locus (QTL)
Using a syntenic approach through the UCSC genome browser LiftOver tool, we observed a putative orthologous Alivec locus in the human genome on chromosome 15, adjacent to ACAN ( Figure 8A) [40]. This locus consists of an expressed sequence tag (EST) BF961603, that may represent the human ortholog, ALIVEC ( Figure 8A). This EST is 514 nucleotides in size and does not have coding potential with a probability of 0.00511, as assessed by CPC2 analysis. Similar to the rat Alivec locus, the human locus has H3K27ac peaks (shaded region) that are predicted to be an ACAN-regulating enhancer element based on GeneHancer (GeneCards) regulatory elements in the UCSC genome browser. This enhancer locus has a higher PhyloP conservation score, suggesting a similarity between the rat and human genome [41]. The upstream genomic region of ALIVEC-EST possesses a SOX9-binding site similar to the Alivec upstream region in rats. Interestingly, the human ALIVEC locus is part of a QTL associated with blood pressure, identified from genetic analysis of inherited hypertension in rats and by further genome lift-over to human genomes [42]. It harbors six expression QTLs (eQTLs) that regulate ACAN expression ( Figure 8A) [43]. Moreover, we found that AngII-treated human VSMCs (HVSMCs) showed increased expression of ALIVEC ( Figure 8B) and ACAN ( Figure 8C), relative to controls. These results suggest that the putative human ALIVEC gene locus and transcript have similar features and functions as the rat Alivec. mRNA and H19 lncRNA (negative controls, Figure 6C). In addition, mass spectrometry showed that Alivec interacts with the RNA-binding protein, heterogeneous nuclear ribo-nucleoproteinA2B1 (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. Figure 6. Alivec RNA interacts with hnRNPA2B1 and tropomyosin-3-alpha, a protein potentially associated with the contractile phenotype of VSMCs. (A) Network of protein complexes generated (using STRING database) from Alivec-specific interacting proteins identified by RNA-pulldown coupled to mass spectrometry. (B) Western blot analysis with Tpm3 antibody (upper panel) or hnRNPA2B1 antibody (lower panel) following RNA-pulldown assays with RVSMCs extracts using biotinylated Alivec RNA and poly A RNA as negative control (Control). (C) RNA immunoprecipitation assays with UV-cross-linked RVSMC cell extracts using anti-Tpm3 antibody and IgG as negative control. RNA from Tpm3 and IgG immunoprecipitates were analyzed by RT-qPCR, using indicated primers. Results were shown as fold enrichment over IgG. Data presented as mean ± SD, n = 3 biological replicates and ** p < 0.01 vs. IgG, using unpaired Student's t-test. N.s. indicates not significant. Figure 6. Alivec RNA interacts with hnRNPA2B1 and tropomyosin-3-alpha, a protein potentially associated with the contractile phenotype of VSMCs. (A) Network of protein complexes generated (using STRING database) from Alivecspecific interacting proteins identified by RNA-pulldown coupled to mass spectrometry. (B) Western blot analysis with Tpm3 antibody (upper panel) or hnRNPA2B1 antibody (lower panel) following RNA-pulldown assays with RVSMCs extracts using biotinylated Alivec RNA and poly A RNA as negative control (Control). (C) RNA immunoprecipitation assays with UV-cross-linked RVSMC cell extracts using anti-Tpm3 antibody and IgG as negative control. RNA from Tpm3 and IgG immunoprecipitates were analyzed by RT-qPCR, using indicated primers. Results were shown as fold enrichment over IgG. Data presented as mean ± SD, n = 3 biological replicates and ** p < 0.01 vs. IgG, using unpaired Student's t-test. N.s. indicates not significant. 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). (v) proteins on aortic tissue sections from vehicle or AngII-infused rats, scale bar: 50 µ M. Box plots on the right show the quantification of aortic staining of indicated proteins shown in panels (ii) to (v). Box plots on the right show integrated density (IntDen) expressed as fold-over control. Staining was quantified using ImageJ software in 20 different areas for each group (3 aortas in control and 3 in AngII group. Data represented as mean and minimum/maximum, unpaired Student's t-test and *** p < 0.001 and **** p < 0.0001). (C-E) RT-qPCR analysis showing gene expression of Alivec, Acan and Runx1 in aortas from AngII-infused rats in comparison to vehicle-treated rats. Data presented as mean ± SD, n = 3 biologic replicates and unpaired Student's t-test. * p < 0.05 vs. vehicle.

Discussion
LncRNAs are important regulators of VSMC function and play key roles in the development of CVDs [16,23]. Hypertensive animals show loss of a VSMC contractile phenotype and gain of a chondrogenic phenotype [44]. This study is the first to identify a lncRNA (Alivec) that regulates the expression of the AngII-induced genes known to be involved in the trans-differentiation of VSMCs to chondrocyte-like cells. We found that Alivec and its neighboring protein-coding gene Acan (usually associated with chondrocytes) are induced by AngII as well as by other stimuli known to promote VSMC de-differentiation. Furthermore, in RVSMCs, AngII induces Alivec through AT1R via Src and ERK1/2 and TF Sox9. Gain of function and knockdown approaches clearly demonstrated that Alivec can modulate AngII-induced chondrogenic genes. Altogether, our study suggests a role for Alivec in promoting the chondrogenic differentiation of VSMCs.
Aggrecan (Acan), also known as chondroitin sulfate proteoglycan 1, is encoded by the Acan gene (ACAN in humans) [45,46]. Acan protein is an integral part of the extracellular matrix in cartilage. It is increased with vascular injury and is associated with hypertension [47,48]. TNF-α-induced protein-6 (TSG-6), a protein product of TNFAIP6, and ACAN protein interact to promote matrix stability [49]. The oxidized low-density lipoprotein receptor 1 (OLR1), also known as LOX1, has a pro-inflammatory role. Olr1 is induced by AngII and is associated with phenotypic changes in VSMCs and hypertension [50,51]. Osteopontin, a multifunctional glycophosphoprotein protein, is encoded by the SPP1 gene (secreted phosphoprotein 1). Osteopontin is produced by VSMCs, endothelial cells and macrophages, and plays an important role in physiological and pathophysiological processes, including the regulation of biomineralization and VSMC calcification [52][53][54]. The role of lncRNAs in regulating these genes in VSMCs is not known. We found that the knockdown of Alivec led to a reduction of AngII-induced Alivec and Acan, as well as the chondrocyte-associated genes Tnfaip6, Runx1, Olr1 and Spp1. Furthermore, as indicated by alcian blue staining, AngII treatment promoted a chondrocyte-like phenotype in VSMCs, which was attenuated with Alivec knockdown and increased by Alivec overexpression, implicating a functional role for Alivec in chondrogenic transformation of VSMCs.
Moreover, aortas from AngII-treated hypertensive rats displayed increased expression of Alivec and Acan along with reduced expression of contractile genes. These findings suggest that Alivec upregulation occurs under conditions of hypertension and aortic wall remodeling. Relevant to this, VSMCs, chondrocytes and osteoblasts are all derived from mesenchymal stem cells [55]. Moreover, arterial injury in SM22 knockout mice resulted in VSMC transformation to chondrocyte-like cells [31]. In light of these new and published findings, rodent models of atherosclerosis and chronic hypertension may reveal unique interactions between plaque formation, arterial remodeling, vascular calcification and Alivec.
Our examination of lncRNA-binding proteins provided key insights into the mechanisms of action and regulation of the target genes by Alivec in VSMC. We previously showed that interaction of AngII-induced lncRNA Giver with the RNA-binding protein NONO played a role in regulating genes associated with inflammation and oxidative stress in VSMCs [23]. Herein, mass spectrometry analyses of RNA-pulldown experiments identified Tpm3 and hnRNPA2B1 as major Alivec interacting proteins. STRING analysis of these and other Alivec interacting protein-binding partners provided clues regarding potential mechanisms, through which Alivec regulates target gene expression and enhances the chondrocyte phenotype of VSMCs. Tropomyosins are cytoskeletal proteins that regulate smooth muscle cell contraction through interaction with actin. Levels of tropomyosin 1 (Tpm1) protein were downregulated in response to high glucose in VSMCs, and this augmented VSMC transition to a synthetic phenotype [56,57]. It is possible that AngII, by increasing cytosolic Alivec, could sequester Tpm3 and inhibit its functions, leading to reduction in the contractile features of VSMCs, while increasing their synthetic and chondrogenic features. Concurrently, nuclear Alivec, through interactions with hnRNPA2B1, might regulate other target genes in trans, including chondrogenic genes. Alivec overlaps an enhancer, suggesting it could potentially be an enhancer-RNA (eRNA) and may also regulate the neighboring gene Acan through enhancer activity. But further in-depth studies are needed to determine the enhancer effects of the Alivec locus and Alivec's function as eRNA in VSMCs. Spp1 is a target gene of Alivec that we identified and hnRNPA2B1 is involved in the regulation of Spp1 expression in macrophages [58]. Similar to Alivec, lincRNA-Cox2 is localized in the nuclear and cytoplasmic compartments of macrophages [59]. Nuclear lincRNA-Cox2 interacts with hnRNPA2B1 and regulates the expression of immune genes in response to activation of toll-like receptor signaling [59]. Together these data suggest that Alivec acts via nuclear hnRNPA2B1 and cytoplasmic Tpm3 to alter gene expression and phenotype. However, additional mechanistic studies, including determining the direct functions of Tpm3 and hnRNPA2B1 in VSMCs, are needed to confirm this.
Of translational relevance, we identified a potential human ortholog of ALIVEC in AngII-treated HVSMCs. Interestingly, this ALIVEC locus is part of a QTL associated with blood pressure. Identification of this QTL was based on the genetic analysis of inherited hypertension in rats and by further genome lift-over to humans [42]. However, the function of these variants and their association with human hypertension, has not been determined. In addition, ATAC-seq data from the transforming growth factor (TGF)-β-treated human coronary artery SMCs, identified an inducible open chromatin region in the enhancer region of the ALIVEC locus (Supplementary Figure S4) [60]. These data suggest, similar to the rat locus, the presence of an active enhancer element in the ALIVEC locus of the human genome that is responsive to TGF-β and PDGF. Furthermore, the presence of open chromatin in this region, along with the H3K27ac peak predicted as an ACAN regulating enhancer, supports connections between ALIVEC, VSMC chondrogenic-like phenotype and blood pressure. Furthermore, an EST in this region was also induced by AngII in HVSMCs. However, additional studies are needed to fully characterize the putative orthologous human transcript and determine its potential connections to human hypertension.
Limitations of the study include the paucity of details on how Alivec-interacting proteins modulate VSMC function, as well as the inadequate characterization of the putative human transcript and the functional relationship to AngII-induced hypertension. Additional mechanistic studies are required to elucidate the nuclear vs. cytoplasmic actions of lncRNA Alivec. Examination of remodeled and calcified arteries of hypertensive rodents and humans may help in identifying Alivec as a potential biomarker for CVD development and progression. Nonetheless, the data presented demonstrate that a novel AngII-induced lncRNA Alivec regulates genes associated with the VSMC phenotypic transition to chondrocyte-like cells and is likely associated with blood pressure regulation. These results provide new insights into the role of lncRNAs in chondrogenesis and key pathologic vascular actions of AngII that could lead to new therapeutic targets for AngII-regulated CVDs.

Conclusions
Together these results demonstrate that a novel AngII induced lncRNA Alivec regulates genes associated with chondrogenic transformation of VSMCs implicated in vascular dysfunction, which could lead to the identification of non-coding RNA based biomarkers and therapeutic targets for CVDs.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/cells10102696/s1, Figure S1: Alivec characterization and full-length cloning. Figure S2: Design and efficacy of LNA-GapmeRs targeting Alivec. Figure S3: Microarray profiling in RVSMCs transfected with NCGap or AlivecGap. Figure S4: Chromatin accessibility at the putative human ALIVEC locus in human coronary artery smooth muscle cells (HCASMCs). Table S1: Primer sequences used in the study. Table S2: Complete Alivec sequence. Table S3: GapmeRs and siRNA sequences. Table S4: Antibodies used in the study and their source.