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LncRNA PSR Regulates Vascular Remodeling Through Encoding a Novel Protein Arteridin

Originally publishedhttps://doi.org/10.1161/CIRCRESAHA.122.321080Circulation Research. 2022;131:768–787

Abstract

Rationale:

Vascular smooth muscle cells (VSMCs) phenotype switch from contractile to proliferative phenotype is a pathological hallmark in various cardiovascular diseases. Recently, a subset of long noncoding RNAs was identified to produce functional polypeptides. However, the functional impact and regulatory mechanisms of long noncoding RNAs in VSMCs phenotype switching remain to be fully elucidated.

Objectives:

To illustrate the biological function and mechanism of a VSMC-enriched long noncoding RNA and its encoded peptide in VSMC phenotype switching and vascular remodeling.

Results:

We identified a VSMC-enriched transcript encoded by a previously uncharacterized gene, which we called phenotype switching regulator (PSR), which was markedly upregulated during vascular remodeling. Although PSR was annotated as a long noncoding RNA, we demonstrated that the lncPSR (PSR transcript) also encoded a protein, which we named arteridin. In VSMCs, both arteridin and lncPSR were necessary and sufficient to induce phenotype switching. Mechanistically, arteridin and lncPSR regulate downstream genes by directly interacting with a transcription factor YBX1 (Y-box binding protein 1) and modulating its nuclear translocation and chromatin targeting. Intriguingly, the PSR transcription was also robustly induced by arteridin. More importantly, the loss of PSR gene or arteridin protein significantly attenuated the vascular remodeling induced by carotid arterial injury. In addition, VSMC-specific inhibition of lncPSR using adeno-associated virus attenuated Ang II (angiotensin II)–induced hypertensive vascular remodeling.

Conclusions:

PSR is a VSMC-enriched gene, and its transcript IncPSR and encoded protein (arteridin) coordinately regulate transcriptional reprogramming through a shared interacting partner, YBX1. This is a previously uncharacterized regulatory circuit in VSMC phenotype switching during vascular remodeling, with lncPSR/arteridin as potential therapeutic targets for the treatment of VSMC phenotype switching–related vascular remodeling.

Novelty and Significance

What is Known?

  • Vascular smooth muscle cell (VSMC) phenotype switching is a core pathological process in vascular remodeling.

  • Long noncoding RNAs (lncRNAs) can regulate key aspects of vascular pathology.

  • A subset of lncRNAs is identified to produce functional polypeptides

What New Information Does This Article Contribute?

  • A novel VSMC-enriched lncRNA, phenotype switching regulator (PSR), is upregulated during vascular remodeling and is necessary for VSMC phenotype switching and neointima formation.

  • Although annotated as lncRNA, PSR encodes a 117aa functional protein, arteridin.

  • arteridin and lncPSR noncoding transcript produced from the PSR gene work coordinately in inducing VSMC phenotype switching. They directly interact with and regulate downstream genes through a transcription factor YBX1 (Y-box binding protein 1) by modulating its nuclear translocation and chromatin targeting, respectively. PSR transcription is also robustly induced by arteridin, forming a positive feedforward loop.

VSMC phenotype switching is associated with vascular remodeling in various cardiovascular diseases. Recently, a subset of lncRNAs was identified to produce both polypeptides and functional regulatory transcripts. Here, we identified a VSMC- enriched lncRNA named PSR, which was markedly upregulated during vascular remodeling. Although annotated as a lncRNA, PSR encodes a protein, arteridin. In VSMCs, both arteridin and lncPSR were necessary and sufficient to induce phenotype switching from a contractile to a proliferative phenotype. Furthermore, arteridin and lncPSR regulated downstream genes through direct interaction with YBX1, a transcription factor implicated in neointimal hyperplasia and vascular remodeling, by modulating its nuclear translocation and chromatin targeting. In vivo, ablation of PSR gene or arteridin protein significantly attenuated carotid arterial remodeling induced by balloon injury. VSMC-specific inhibition of lncPSR also attenuated Ang II–induced vascular remodeling associated with hypertension. Finally, the expression of lncPSR and arteridin protein was significantly altered in human mesenteric arteries associated with pathological remodeling. Together, our study provides a previously uncharacterized regulatory complex in VSMC phenotype switching during vascular remodeling and suggests that lncPSR/arteridin may serve as potential therapeutic target for the prevention of vascular remodeling.

Meet the First Author, see p 727

Vascular remodeling is a major pathological feature associated with chronic hypertension or other vascular injuries that contributes to cardiovascular complications, including myocardial infarction, stroke, atherosclerosis, and restenosis after stent placement.1,2 Vascular smooth muscle cells (VSMCs) are key players in vascular remodeling and subsequent dysfunction.3,4 They are maintained in a more differentiated contractile state in healthy vessels. In response to pathological injury, however, VSMCs switch to a more de-differentiated phenotype characterized by induction of synthetic and proliferative activities.3,5 This VSMCs phenotype switching is central to vascular wall thickening, stenosis, and other pathological changes during pathological remodeling.5 Understanding the regulatory mechanisms that govern phenotype switching in VSMCs holds major promise for better diagnosis and treatment of vascular diseases.

Following the complete sequencing of the human genome, the human transcriptome complexity has dramatically expanded to include large numbers of long noncoding RNAs (lncRNAs).6 However, there is an emerging recognition that a subset of the annotated lncRNAs (roughly 5%) in the human genome contains an open reading frame (ORF) that has the translational capacity to produce functional proteins/peptides.7-9 Whether these peptides truly exist and what roles they play in the cardiovascular system remain largely unknown. In the currently characterized regulatory network of VSMC phenotype switching-regulated vascular remodeling, protein/peptide encoded by an annotated lncRNA has not been reported.

From an unbiased transcriptome analysis in the aorta tissue of the spontaneously hypertensive rat (SHR), we identified a previously uncharacterized VSMCs abundant lncRNA, now named as phenotype switching regulator (PSR), and identified a 117 amino acid peptide arteridin encoded by lncPSR. We found that arteridin induces VSMC phenotype switching through direct interaction with a transcription factor YBX1 (Y-box binding protein 1), and this interaction also requires lncPSR transcript. arteridin-YBX1-lncPSR complex regulates the expression of phenotype switching–related genes in VSMCs by modulating YBX1 nuclear translocation and chromatin targeting. Moreover, germline ablation of PSR gene or arteridin protein reduced balloon injury-induced neointima formation in the carotid artery in rats. In addition, VSMC-specific knockdown of lncPSR using an adeno-associated virus (AAV) based system attenuated the Ang II (angiotensin II) induced vascular remodeling in mice. In summary, our findings uncovered a previously uncharacterized genetic regulatory circuit in VSMCs reprogramming, involving a lncRNA and its encoded protein. This regulatory network can serve as potential therapeutic target for the treatment of pathological vascular remodeling.

Methods

Data Availability

All the data supporting the findings of this study are available within the article and its Supplemental Material. The chromatin immunoprecipitation–sequencing and lncRNA-mRNA microarray datasets are deposited into Gene Expression Omnibus (GEO) with accessing code: GSE 145965.

Animal

All experimental procedures involving animals in this study were approved by the Commit of Animal Research Institute of The Third Military Medical University, China, and were conducted conforming with the US National Institutes of Health guidelines for the care and use of laboratory animals. Detailed Materials and Methods are provided in the Supplemental Material.

Results

VSMC-Enriched lncRNA PSR Is Upregulated During Vascular Remodeling

To gain a better insight into the underlying molecular changes during vascular remodeling, we applied transcriptomic analysis using an mRNA-lncRNA microarray on thoracic aortae from 24-week-old spontaneously hypertensive rats (SHR), which is an established model of hypertension-induced vascular remodeling,10,11 and normotensive Wistar-Kyoto rats as controls (differentially expressed lncRNAs shown in Figure 1A and differentially expressed mRNAs shown in Figure S1A). A subset of the identified differentially expressed lncRNAs (Table S1) was further validated by qRT-PCR (quantitative real-time PCR) (Figure 1B). Among them, the candidate lncRNA NR_027983 (ENSRNOT00000080801.1, ENSEMBL (genome database) Rnor_6.0; LOC680254 gene) had an abundant aortic expression profile at baseline in adult Sprague Dawley (SD) rats (Figure 1C). We named this putative lncRNA phenotype switching regulator (PSR) due to its apparent role in VSMCs phenotype switching as revealed in the subsequent studies. The full-length lncPSR transcript of 890 nt was detected by Northern blot in multiple tissues from adult SD rat, which is consistent with the sequence information in ENSEMBL database (Figure 1D and Figure S1B). LncPSR (PSR transcript) has a relatively enriched expression in aorta (Figure 1D), although significant levels of expression were also detected in other tissues at baseline. Using RNAscope in situ hybridization, lncPSR expression was found to be highly enriched in the tunica media of the vascular wall colocalizing with VSMCs (Figure 1E). This VSMC predominant expression was also confirmed by qRT-PCR using primary cultures of VSMCs, fibroblasts, and endothelial cells (Figure S1C). In VSMCs, lncPSR was detected mainly distributed in the cytoplasm as determined by RNAscope fluorescence in situ hybridization and qRT-PCR after subcellular fractionation (Figure 1F and 1G). Moreover, the LncPSR gene was conserved among rat, mouse (2810025M15Rik, NR_027984), and human (RASAL2-AS1 [RASAL2 antisense RNA 1], NR_027982) based on both RNA sequences and genomic positions (Figure S1D).

Figure 1.

Figure 1. Long noncoding RNA (LncRNA) phenotype switching regulator (PSR) is abundantly expressed in vascular smooth muscle cells (VSMCs) and is upregulated during vascular remodeling. A, Identification of differentially expressed long noncoding RNAs (lncRNAs) associated with hypertension induced vascular remodeling in rats. Heatmap depicts levels and identities of the significantly differentially expressed lncRNAs (spontaneously hypertensive rat [SHR] vs Wistar-Kyoto (WKY), fold change>1.2 or <0.8, p<0.05, false discovery rate<0.05) detected by microarray analysis in the aortic tissues from 24-wk-old WKY and SHR rats (n=3). The log2 fold change represents the ratio of base mean (the average expression levels of samples were compared after standardization) between SHR and WKY rats. The redder color of the log2 fold change indicates higher level of gene expression. B, qRT-PCR (quantitative real-time PCR) validation of candidate differentially expressed lncRNAs screened out from lncRNA-mRNA microarray in aortae tissue of 24-wk-old WKY rats and SHRs. The relative expression levels are normalized to CYPA (cyclophilin A). The results for ENSRNOT00000016107 and ENSRNOT00000070662 are presented as median±interquartile, vs WKY, by Mann-Whitney U test. The results for the other lncRNAs are presented as mean±SEM, vs WKY, by unpaired Student t test (n=6). C, Tissue-specific expression pattern as detected by RT-PCR for the top candidate lncRNAs with differential expression in aortic tissues between 24-wk-old WKY and SHR rats. The signal for CYPA mRNA is used as a normalization control. Total RNA was extracted from different tissues of adult Sprague Dawley (SD) rats as indicated. D, Northern blot of adult SD rat tissues showing lncPSR expression. 18S and 28S rRNAs are visualized as loading controls. E, LncPSR expression in adult SD rat aorta tissue detected by RNAscope in situ hybridization. Rat Polr2a mRNA is used as a positive control. A nontarget probe is used as a negative control (NC). Arrows indicate positive RNA signals. Scale bar: 200 μm. F, Intracellular localization of PSR transcripts in rat primary VSMCs. RNA transcripts are detected by RNAscope-fluorescent in situ hybridization (red) and nuclei are labeled by DAPI (4'‚6-diamidino-2-phenylindole) (blue) in primary VSMCs isolated from adult SD rats. Polr2a (RNA Polymerase II Subunit A) mRNA was detected as a positive control. A nontarget probe is used as an NC. Arrows indicate positive RNA signals. Scale bar: 20 μm. G, Intracellular distribution of lncPSR in rat aortic VSMCs detected by qRT-PCR. GAPDH is used as a cytoplasmic control, and Snora41 is used as a control for nuclear targeted transcript. Data are mean±SEM performed in triplicates. H, Expression of lncPSR in the aortae tissue of 4, 14, and 24 weeks old of WKY and SHR rats detected by qRT-PCR. The relative expression levels at different time points are normalized to signal from 4 wk old WKY rats after first normalized to CYPA. Data are median±interquartile range, vs WKY, by Mann-Whitney U test (n=5). I, Expression of lncPSR in the carotid artery tissue of adult SD rats 28 d after sham or balloon injury detected by qRT-PCR, normalized to CYPA. Data are mean±SEM, vs sham, by unpaired Student t test (n=7). J, PSR expression in adult SD rat carotid artery tissue 14 d after sham (top) or balloon injury (bottom) detected by RNAscope in situ hybridization (red), coimmunostained with VSMC marker protein α-SMA (α-smooth muscle-actin; green), and nuclei were labeled by DAPI (blue). Scale bar: 100 μm. K, Relative expression levels of human lncPSR detected with qRT-PCR in healthy mesenteric arteries (HMA) and remodeled mesenteric arteries (RMA), normalized to GAPDH. Data are mean±SEM, vs HMA, by unpaired Student t test (n=8). A indicates adventitia; M, media; N, neointima; and sk. muscle, skeletal muscle.

The transcript level of lncPSR was significantly upregulated in the aorta in the 24-week-old hypertensive SHRs compared with the normotensive Wistar-Kyoto controls (Figure 1H). However, lncPSR expression was not increased in the aortic tissue in 4-week-old SHRs, before the onset of hypertension, or in the aorta of 14-week-old SHRs when there is no notable vascular remodeling despite the presence of hypertension (Figure 1H). Consistent with this result, the expression level of lncPSR was also markedly upregulated in the rat carotid artery after balloon injury which is a potent inducer of vascular remodeling (Figure 1I).12,13 Notably, RNAscope of lncPSR and α-SMA (α-smooth muscle-actin) immunofluorescent costaining showed that upregulated lncPSR was mainly in the VSMCs in rat carotid arteries with balloon injury (Figure 1J).

To further evaluate the potential clinical implication of our findings, we collected mesenteric arteries from gastrointestinal nontumor tissue adjacent to tumors from patients who underwent radical resection of gastrointestinal cancer and divided the samples into two groups based on histological evidence with or without vascular remodeling. The basic clinical information of the donors is described in Table S2. As shown in Figure 1K, and Figure S1E, human lncPSR expression was significantly higher in the remodeled mesenteric arteries (RMA) compared with the nonremodeled healthy mesenteric samples (HMA) (Figure 1K). Taken together, these results indicate that lncPSR is a VSMC-enriched transcript and its expression is dynamically regulated in association with pathological vascular remodeling in both rodents and humans.

LncPSR Is Necessary for VSMCs Phenotype Switching In Vitro and In Vivo

The potential role of lncPSR in VSMC phenotype switching was first investigated in rat primary aortic VSMCs. Treatment with Ang II (angiotensin II) increased, whereas TGF (transforming growth factor)-β decreased lncPSR expression in VSMCs (Figure 2A and 2B). Knockdown of lncPSR in VSMCs using small interfering RNA (Figure S2A and S2B) significantly induced the baseline mRNA and protein expressions of marker genes associated with the contractile state, including actin alpha 2, smooth muscle (ACTA2; encoding α-SMA), calponin 1 (CNN1), and transgelin (TAGLN; encoding SM22 [smooth muscle protein 22]; Figure 2C and 2D). The maintenance of a contractile phenotype by lncPSR knockdown was also observed in human aortic smooth muscle cells (Figure 2C and 2D). Along with the impact on marker gene expression, lncPSR silencing significantly reduced rat primary aortic VSMC proliferation and migration at basal, as well as in response to PDGF (platelet-derived growth factor)-BB treatment (Figure 2E through 2H), supporting its necessary role in VSMCs phenotype switching, proliferation, and migration in vitro.

Figure 2.

Figure 2. Phenotype switching regulator (PSR) is necessary for vascular smooth muscle cells (VSMCs) phenotype switching in vitro. A, Expression of lncPSR in primary VSMCs treated with Ang II (angiotensin II, 0.1 μmol/L) at different time points detected by qRT-PCR (quantitative real-time PCR), normalized to CYPA (cyclophilin A). Data are median±interquartile range, vs control, Kruskal-Wallis test with Dunn post hoc analysis (n=4). B, Expression of lncPSR in primary VSMCs treated with TGF (transforming growth factor)-β (10 ng/mL) detected by qRT-PCR, normalized to CYPA. Data are median±interquartile range, vs negative control (NC), by Mann-Whitney U test (n=4). C, Relative mRNA levels detected by qRT-PCR of VSMCs contractile markers, ACTA2, CNN1, and TAGLN genes in rat aortic VSMCs treated with scramble small interfering RNA (siRNA) or siPSR (small interfering RNA targeting lncPSR) for 24 h, normalized to CYPA. Data are median±interquartile range, vs NC, by Mann-Whitney U test (n=4). D, Representative Western blot for protein levels of VSMCs contractile markers α-SMA (α-smooth muscle-actin), Calponin, and SM22 (smooth muscle protein 22) in rat aortic VSMCs treated with scramble siRNA or siPSR for 48 h. GAPDH was used as a loading control (top). Quantification of protein levels normalized against GAPDH (bottom). Data are median±interquartile range, vs NC, by Mann-Whitney U test (n=5). E, Representative immunofluorescent images of EdU (5-ethynyl-2’-deoxyuridine) (red), and nuclei (DAPI, blue) in rat primary VSMCs transfected with lentiviruses harboring scramble sequence (lenti-scramble) or shPSR (short hairpin RNA targeting lncPSR) (lenti-shPSR) at baseline or under PDGF (platelet-derived growth factor)-BB treatment (25 ng/mL) for 48h (left). Scale bar: 100 μm. Quantification of percentage of EdU positive VSMCs transfected with lenti-scramble or lenti-shPSR lentivirus at baseline or under PDGF-BB (25 ng/mL) treatment. Data are mean±SEM, vs lenti-scramble, by 2-way ANOVA with Sidak post hoc analysis (n=6, 3 random visual fields from each sample). F, Proliferation activity measured by Cell Counting Kit 8 (CCK-8) for isolated rat aortic VSMCs transfected with lent-scramble or lenti-shPSR with PDGF-BB (25 ng/mL) treatment for 24 h, 48 h, and 72 h. Proliferation rate is normalized to OD450 at 0 h time point post–PDGF-BB treatment. Data are median±interquartile range, vs lenti-scramble, by Mann-Whitney U test (n=4). G, Representative images of in vitro scratch wound healing assays using isolated VSMCs transfected with lenti-scramble or lenti-shPSR for 24 h followed by PDGF-BB (25 ng/mL) treatment for 12 and 24 h as indicated. Lines depict edges of cell sheet in culture (left). Scale bar: 100 μm. Quantification of cell migration based on wound healing assay (right). Surface of scratching was measured at time 0, 12, and 24 h after PDGF-BB treatment, and migration indexes are quantified based on percentage of surface area changes at 12 and 24 h post treatment relative to the surface area at time 0 as indicated. Data are mean±SEM, vs lenti-scramble, by 2-way ANOVA with Sida post hoc analysis (n=6). H, Representative images of trans-well migration assays using isolated VSMCs transfected with lenti-scramble or lenti-shPSR after PDGF-BB (25 ng/mL) treatment for 12 h (left). Scale bar: 100 μm. The quantification is indicated on the right. Migrated cells are counted and calculated as fold change after normalized to the migrated cell number with the lenti-scramble transfected VSMCs. Data are median±interquartile range, vs lenti-scramble, by Mann-Whitney U test (n=4).

To determine the in vivo function of PSR gene, we ablated the PSR gene in the rat (PSR–knock-out [KO]) by CRISPR/Cas9-mediated deletion of the entire locus (Figure S2E and S2F). Complete loss of lncPSR transcript was validated by qRT-PCR (Figure S2G). The PSR-KO rats had normal viability and development with no changes in baseline blood pressure (Figure 2H through 2J). However, following carotid artery balloon injury, the neointima formation was significantly attenuated in the PSR-KO rats compared with the wild-type (WT) controls (Figure 3A through 3C). Immunofluorescence staining revealed higher expression of α-SMA in the PSR-KO rat at baseline. The expression of α-SMA was also preserved after injury in the PSR-KO rat (Figure 3D), accompanied by higher expression of contractile marker genes (Figure 3E) and reduced Ki-67 positive VSMCs in the postinjury carotid arteries (Figure 3F). These results provided clear in vivo validation that the PSR gene is a necessary regulator of VSMCs phenotype switching during vascular remodeling.

Figure 3.

Figure 3. Phenotype switching regulator (PSR) is necessary for vascular smooth muscle cells (VSMCs) phenotype switching in vivo. A, Representative hematoxylin and eosin staining of left carotid arteries (LCA, bottom) from a wild-type (WT) and a PSR knock-out (KO) rat at 14 d after balloon injury. The right carotid arteries (RCA) from the same rats are analyzed as the corresponding sham controls (top). Scale bar: 100 μm. B, Quantification of intima area. Data are mean±SEM, vs WT, by unpaired Student t test (n=7). C, Quantification of neointima-to-media ratio. Data are mean±SEM vs WT, by unpaired Student t test (n=7). D, left, Representative images of immunofluorescence staining of α-SMA (α-smooth muscle-actin; green) and nucleic staining DAPI (blue) of carotid arteries (LCA, bottom) from WT and PSR-KO rats at 14 d after balloon injury. The RCA from the same rats were imaged as the sham controls (top). Scale bar: 100 μm. Right: Quantification of α-SMA immunofluorescence staining integrated density of RCA (sham) or LCA (injury) from WT and PSR-KO rats at 14 d after balloon injury. Data are mean±SEM, vs WT, by 2-way ANOVA with Sidak post hoc analysis (n=6). E, Top, Representative Western blot for protein levels of VSMCs contractile markers α-SMA, calponin, and SM22 (smooth muscle protein 22) in LCA from WT and PSR-KO rats at 14 d after balloon injury or the RCA from the same rats as the sham controls. Bottom, Quantification of Western blot for VSMCs contractile markers α-SMA, calponin, and SM22 in LCA from WT and PSR-KO rats at 14 d after balloon injury or the RCA from the same rats as the sham controls, normalized to GAPDH (n=6 rats/group). Data are mean±SEM, vs WT sham or WT injury as indicated, by 2-way ANOVA with Sidak post hoc analysis (n=6). F, left, Representative immunofluorescent images of Ki-67 staining (red) and α-SMA (green) in LCA from WT and PSR-KO rats at 14 d after balloon injury. DAPI (blue) was used for nucleic labeling. Scale bar: 100 μm. Right, Qualification of the Ki-67-positive VSMCs presented as percentage of the total number of VSMCs. Data are mean±SEM, vs WT, by unpaired Student t test (n=7). G, Heatmap depicting differentially expressed genes (DEGs) in rat A10 thoracic artery VSMCs transfected with scramble small interfering RNA (siRNA) or siRNA targeting lncPSR detected with RNA-seq (RNA sequencing; vs scramble, n=3). The Log2 fold change represents the ratio of basemean (the average expression levels of samples were compared after standardization) between siPSR and scramble siRNA. The redder color of the Log2 fold change indicates higher level of gene expression. H, Heatmap depicting expression change of a subset of DEGs relevant to the phenotype of VSMCs identified from the RNA-seq dataset (vs scramble, n=3). The Log2 fold change represents the ratio of basemean (the average expression levels of samples were compared after standardization) between siPSR and scramble siRNA. The redder color of the Log2 fold change indicates higher level of gene expression. I, Significant positive correlation (normalized enrichment score [NES]>1, false discovery rate [FDR]<0.05) of genes altered by lncPSR knockdown with Negative regulation of smooth muscle cell proliferation (left) and smooth muscle cell contraction (right) in VSMCs as determined by Gene Set Enrichment Analysis. A indicates adventitia; GOBP, Gene Ontology Biological Process; M, media; and N, neointima.

To further determine the impact of lncPSR on VSMCs phenotype regulation, we performed global transcriptomic analysis in rat A10 thoracic aorta vascular smooth muscle cells, undifferentiated VSMCs as an established in vitro model for VSMCs differentiation.14-16 As shown in Figure 3G, the VSMCs transcriptome was significantly altered by lncPSR knockdown, including many VSMC phenotype related genes (Figure 3H). Gene Ontology Biological Process analysis showed that genes affected by lncPSR knockdown were enriched in pathways of extracellular matrix organization, vascular development, cell proliferation, and migration (Figure S2K), consistent with its role in phenotype switching regulation.1,17 Gene set enrichment analysis further revealed that the genes altered by lncPSR knockdown had significant positive correlation with smooth muscle contraction and negative regulation of smooth muscle cell proliferation (Figure 3I), all supporting the impact of lncPSR knockdown on VSMCs differentiation.

Identification of Arteridin Protein Encoded by lncPSR

The PSR gene was originally annotated as a lncRNA. However, five potential ORFs are present in the rat PSR transcript, with the longest one encoding a putative protein containing 117 amino acids with 92% amino acid sequence identity between rat and mouse (Figure S3A and S3B). Notably, this putative 117aa (117 amino acids) protein has now been re-annotated as RCG46462 and A0A0G2JYX1 in updated Uniprot and ENSEMBL databases, respectively. Using a polyclonal antibody generated against the predicted 117aa- peptide from the rat ORF, we detected a polypeptide band from adult rat tissues at the expected molecular mass of 12.9 kDa (Figure 4A). Elevated levels of protein signal were readily detected in multiple smooth muscle enriched tissues (Figure 4A), especially in α-SMA positive VSMCs in tunica media (Figure 4B, IgG was set as negative control in Figure S3C). We named this protein product arteridin, due to its initial discovery and functional characterization in arteries. The capacity to produce arteridin protein from lncPSR transcript was further demonstrated in HEK293 (human embryonic kidney 293) cells by the detection of a DYKDDDDK (FLAG)-tagged or an eGFP (enhanced green fluorescent protein)-tagged arteridin fusion protein from the transfected expression vectors containing arteridin ORF cDNAs but not from the vectors containing start codon mutations (Figure 4C, Figure S3D and S3E). These results support the translation capacity of lncPSR and the specificity of anti-arteridin antibody. In agreement with lncPSR expression pattern, arteridin protein expression was also significantly increased in the neointima of carotid arteries after balloon injury in adult SD rats (Figure 4D). Finally, arteridin immunofluorescent signaling was highly enriched in the nucleus of rat aortic VSMCs for either endogenous protein or ectopically expressed FLAG-tagged arteridin (Figure 4E).

Figure 4.

Figure 4. Identification of arteridin protein encoded by long noncoding RNA (lncRNA) phenotype switching regulator (PSR). A, Representative western blot from adult rat tissues using an arteridin-specific antibody, showing the detection of a polypeptide at estimated 12.9 kDa molecular weight. GAPDH is used as a loading control. B, Representative immunofluorescence staining of endogenous arteridin (red), α-SMA (α-smooth muscle-actin; green, labeling smooth muscle cells in the media), and DAPI (blue, labeling nucleus) in adult Sprague Dawley (SD) rat aortic vascular wall. Scale bar: 100 μm. C, Representative western blot using anti- arteridin (left) or anti-FLAG (right) antibodies in HEK293 (human embryonic kidney 293) cells transfected with a plasmid cloning DNA (pcDNA) 5 as negative control‚ an arteridin-FLAG cDNA, or anarteridin-FLAG start codon ATG deletion (arteridin-FLAG ∆ ATG) cDNA as a control. D, Representative immunofluorescence staining of endogenous arteridin (red), α-SMA (green, labeling smooth muscle cells in the tunica media), and DAPI (blue, labeling nucleus) in adult SD rat carotid arteries at 14 d after sham (left) or balloon injury (right). Scale bar: 50 μm. E, Immunofluorescence signals of the arteridin detected using anti- arteridin antibody in primary rat aortic vascular smooth muscle cells (VSMCs) with (red, top) or without (red, bottom) transfected with an adenovirus expressing arteridin-FLAG, costained with DAPI (blue) for nucleic labeling in isolated primary rat aortic VSMCs. IgG is used as negative control. Scale bar: 20 μm. F, Top: Representative hematoxylin and eosin (H&E) staining (upper), and representative immunohistochemistry images (IHC, lower) of arteridin using anti-human arteridin antibody in healthy mesenteric arteries (HMA) or mesenteric arteries with significant remodeling (RMA). Scale bar: 100 μm. Bottom, Quantification of positive IHC signal of arteridin in neointima. Data are median±interquartile range, vs HMA, by Mann-Whitney U test (n=4). A indicates adventitia; M, media; N, neointima; and sk. muscle, skeletal muscle.

In addition to the rat and mouse, an arteridin-like ORF was also identified in the most conserved segment of the human lncPSR transcript which potentially encodes a 106aa peptide (Figure S3F) which is annotated in an updated human proteome dataset hCG1813082 (GenBank: EAW91016.1). Using a newly developed anti-human arteridin antibody with validated specificity (Figure S3G), we found that human arteridin protein was enriched in tunica media from human mesenteric artery tissue (Figure 4F, Figure S3H). Similar to the rat, the arteridin signal was significantly elevated in the proliferating and migrating VSMCs in remodeled neointima in human samples (Figure 4F).

LncPSR, via Arteridin, Regulates VSMCs Phenotype Switching

To determine the in vivo function of arteridin protein in VSMC phenotype switching and vascular remodeling, we generated an arteridin ablation rat (arteridin KO) using CRISPR/Cas9 mediated genome editing to introduce a single T insertion at the 39th nucleotide downstream of the ATG start codon (PSR39_40InsT). This insertion created a premature TAG stop codon (Figure S4A) in the arteridin ORF and resulted in a total loss of the arteridin peptide (Figure 5A), while the level of lncPSR transcript was only modestly reduced (Figure S4B). Like the PSR-KO rats, the arteridin KO rats also showed normal development, viability, and unaltered baseline blood pressure (Figure S4C through S4E). Similar to the PSR-KO rats, neointima formation was also attenuated in the arteridin KO rats following carotid artery balloon injury (Figure 5B through 5D). Immunofluorescence staining showed arteridin KO upregulated α-SMA expression at baseline, and preserved α-SMA expression in postinjury artery (Figure 5E), accompanied by a higher expression of contractile marker genes and reduced staining of Ki-67 positive signal in the VSMCs of carotid arteries (Figure 5F and 5G). All these data support that the arteridin protein is necessary for injury-induced VSMC phenotype switching and vascular remodeling in vivo.

Figure 5.

Figure 5. LncPSR (PSR transcript), via arteridin, regulates vascular smooth muscle cella (VSMCs) phenotype switching. A, Representative Western blot for arteridin peptide from aortic tissue of wild-type (WT) and PSR39_40InsT mutant (arteridin knock-out [KO]‚ by a single T insertion at the 39th nucleotide downstream of the ATG start codon) rats using anti-arteridin antibody (n=3). B, Representative hematoxylin and eosin staining of left carotid arteries (LCA, bottom) from WT and arteridin KO rats at 14 d after balloon injury. The right carotid arteries (RCA, top) from the same rat are used as corresponding sham controls (top). Scale bar: 100 μm. C, Quantification of intimal area from WT and arteridin KO rats 14 d after balloon injury. Data are mean±SEM, vs WT, by unpaired Student t test (n=7). D, Quantification of neointima-to-media ratio from WT and arteridin KO rats 14 d after balloon injury. Data are mean±SEM vs WT, by unpaired Student t test (n=7). E, left, Representative images of immunofluorescence staining of α-SMA (α-smooth muscle-actin; green) and nucleic staining DAPI (blue) of LCA (bottom) from WT and arteridin KO rats at 14 d after balloon injury. The RCA from the same rats are shown as sham controls (top). Scale bar: 100 μm. Right, Quantification of integrated density of α-SMA immunofluorescence signal in RCA (sham) or LCA (injury) from WT and arteridin KO rats at 14 d after balloon injury. Data are mean±SEM, vs WT, by 2-way ANOVA with Sidak post hoc analysis (n=6). F, left, Representative Western blot for protein levels of VSMCs contractile marker genes α-SMA, calponin, and SM22 (smooth muscle protein 22) in LCA from WT and arteridin KO rats at 14 d after balloon injury or the RCA from the same rats as the sham controls. Right, Quantification of Western blot for VSMCs contractile marker genes α-SMA, calponin, and SM22 in LCA from WT and arteridin KO rats at 14 d after balloon injury or the RCA from the same rats as the sham controls, normalized to GAPDH. Data are mean±SEM, vs WT sham or WT injury as indicated, by 2-way ANOVA with Sidak post hoc analysis (n=6). G, left, Representative immunofluorescent images of Ki-67 staining (red) and α-SMA (green) of LCA from WT and arteridin KO rats at 14 d after balloon injury. DAPI (blue) is used for nucleic labeling. Scale bar: 100 μm. Right, Qualification of the Ki-67–positive VSMCs presented as percentage of total number of VSMCs. Data are mean±SEM, vs WT, by unpaired Student t test (n=7). H, Expression of contractile genes detected by qRT-PCR in primary rat aortic VSMCs transfected with lenti-GFP, lenti-phenotype switching regulator (PSR) full-length, lenti-PSR start codon ATG to ATT mutant (ATT mutant), and lenti-arteridin-remix-FLAG, normalized to CYPA (cyclophilin A). Data are median±interquartile range, vs lenti-GFP, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). I, left, Representative Western blots of contractile proteins in primary rat aortic VSMCs transfected with lenti-GFP (lentivirus harhoring green fluorescent protein), lenti-PSR full-length, lenti-PSR full-length ATT mutant, and lenti-arteridin-remix-FLAG. GAPDH was set as loading control. Right, Quantification of Western blot, normalized to GAPDH. Data are median±interquartile range, vs lenti-GFP, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). J, left, Representative Western blots of contractile proteins in primary aortic VSMCs isolated from adult WT or arteridin KO rats, with or without transfected with lenti-GFP or lenti-PSR full-length ATT mutant. GAPDH is a loading control. Right, Quantification of Western blot, normalized to GAPDH. Data are median±interquartile range, vs WT+lenti-GFP, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). A indicates adventitia; M, media; and N, neointima.

The phenocopy between arteridin KO and PSR-KO in rats indicates that arteridin is the functional product of PSR gene, consistent with previously studies that the peptides encoded by annotated noncoding RNAs are the major functional entity.18,19 To further differentiate the potential roles of arteridin versus lncPSR in VSMC phenotype switching regulation, we constructed a vector containing lncPSR full-length transcript but with start codon ATG to ATT mutation at its first codon (referred to as lncPSR full-length ATT mutant). This construct generated lncPSR transcript but without arteridin protein production (Figure S4F). Furthermore, by alternative codon usage, we also constructed a vector containing a synthetic cDNA encoding the same amino acid sequence as the rat arteridin peptide (referred to as arteridin-remix-FLAG) but with more than 30% alterations in the nucleotide sequence. Unexpectedly, the expression of either lncPSR full-length ATT mutant (Figure S4G) or arteridin-remix-FLAG (Figure S4H) all significantly reduced expressions of the contractile genes in rat primary aortic VSMCs, like the full-length lncPSR (Figure 5H and 5I). However, in aortic VSMCs isolated from arteridin KO rats, the effect of lncPSR full-length ATT mutant on phenotype switching was abolished (Figure 5J), indicating that lncPSR transcript, via arteridin protein, regulates VSMCs phenotype switching.

Vascular but Not Hematopoietic Arteridin Is Necessary for Neointima Formation

In addition to vascular cells, bone marrow–derived cells could also contribute to neointima formation.20 Therefore, we further examined the possible contribution of vascular versus hematopoietic arteridin in neointimal formation by mismatched bone marrow transplantation.21 WT and arteridin KO rats were sufficiently irradiated and reconstituted with bone marrow cells derived from either WT or arteridin KO rats (Figure S5A). One week after bone marrow transplantation, we isolated and genotyped the peripheral whole blood cells from the rats to validate the efficiency of bone marrow transplantation (Figure S5B). Balloon injury was performed to induce neointima formation in carotid arteries. The transplantation of bone marrow–derived cells from either WT or arteridin KO rats had neointima areas and intima/media ratios similar to those observed in WT background rats (Figure S5C through S5E). Compared with WT background recipient rats, the recipient rats with arteridin KO background had equally reduced neointima areas and intima/media ratios after receiving WT or arteridin KO bone marrow–derived cells (Figure S5C through S5E). These results excluded the contribution of hematopoietic arteridin in neointimal formation induced by balloon injury, indicating the necessary role of vascularly expressed arteridin in vascular remodeling.

Arteridin Protein and lncPSR Transcript Form a Complex With VSMC Phenotype Switching Inducer YBX1

To explore the molecular basis in arteridin-mediated VSMCs gene regulation, we analyzed the arteridin interacting proteins by immuno-purification of the arteridin complex from rat A10 aortic VSMCs with or without arteridin overexpression, followed by protein identification using mass spectrometry (Figure S6A). For both conditions, the top-ranked molecule identified in the arteridin interacting proteins based on coverage was a transcription factor nuclease-sensitive Element-Binding Protein 1 (also named YBX1; Figure S6B). YBX1 belongs to the cold shock domain-containing gene family with known DNA and RNA binding capacity22-25 and is demonstrated to be a necessary regulator of neointima formation in atherosclerosis-prone ApoE-/- mice.26 Previous studies have reported that YBX1 represses smooth muscle contractile protein gene ACTA2 (α-SMA) through direct binding to the ACTA2 promoter region as a transcription repressor,27,28 as well as interacting with the ACTA2 mRNA reducing the translation efficiency as an RNA binding protein,29,30 suggesting a potential role of YBX1 in the regulation of VSMCs phenotype switching. In rat primary aortic VSMCs, knockdown of YBX1 (Figure S6C and S6D) significantly upregulated the expression of contractile marker genes, while overexpression of YBX1 repressed them (Figure S6E and S6F). We employed lentiviruses expressing short hairpin RNA (shRNA) targeting YBX1 to knockdown YBX1 expression in balloon-injured carotid arteries in 8-week-old SD rats. The neointima formation measured at 28 days postinjury was significantly attenuated in the YBX1 knockdown group compared with the lenti-scramble controls (Figure S6G through S6I), supporting an essential role for YBX1 in postinjury vascular remodeling.

Protein-protein interaction between arteridin and YBX1 was validated by coimmunoprecipitation in HEK293 cells coexpressing rat (Figure 6A) or human (Figure S7A) FLAG-tagged arteridin and hemagglutinin-tagged YBX1. Using various deletion mutants of hemagglutinin-tagged YBX1 (Figure S7B) and coimmunoprecipitation assay in HEK293 cells, the C-terminal domain of YBX1 was found to be responsible for arteridin binding (Figure 6B). In rat aortic VSMCs, the endogenous YBX1 was mainly in the cytoplasm at baseline. However, arteridin overexpression induced a marked translocation of YBX1 from the cytosol to nucleus (Figure 6C and 6D), where arteridin and YBX1 were colocalized (Figure 6C). Immunofluorescent staining showed arteridin KO significantly abolished the nuclear retention of YBX1 in carotid arteries induced by balloon injury (Figure 6E), which was confirmed by subcellular fractionation, followed by Western blotting (Figure 6F). Importantly, we found that silencing of YBX1 abolished the gene expression changes induced by arteridin overexpression (Figure 6G), indicating that YBX1 is a necessary interacting molecule in arteridin-mediated gene regulation and phenotype switching in VSMCs. Using ChIP-qPCR (chromatin immunoprecipitation quantitative real-time PCR), we found that YBX1 occupancies were significantly increased in the promoter of ACTA2 gene in the VSMCs with arteridin overexpression (Figure 6H), further validating that arteridin induces VSMCs phenotype switching through the regulation of the activation of YBX1.

Figure 6.

Figure 6. Arteridin protein and lncPSR (PSR transcript) form a complex with vascular smooth muscle cell (VSMC) phenotype switching inducer YBX1 (Y-box binding protein 1). A, Coimmunoprecipitation (co-IP) of arteridin and YBX1 in HEK293 (human embryonic kidney 293) cells cotransfected with rat arteridin-FLAG and rat YBX1-HA (hemagglutinin tagged YBX1 plasmids) using anti-FLAG or anti-HA antibodies followed by reciprocal immunoblotting (IB) as indicated. B, Schematic drawing of the functional domains of YBX1 (top). Co-IP of arteridin and YBX1 in HEK293 cells cotransfected with arteridin-FLAG with YBX1-HA full-length (wild-type [WT]), N-terminal deletion (del N), C-terminal deletion (del C) and cold shock domain (CSD) deletion followed by Western blot (lower) as indicated. C, Immunofluorescence images of YBX1 in rat aortic VSMCs transfected with control adenovirus (adNull, upper) or arteridin expression adenovirus (ad-arteridin-FLAG, lower) transfection, using anti-YBX1 (green) and anti-arteridin (red) antibodies as indicated. The inserted parts are amplified images of nucleus from YBX1 and arteridin coexpressing cells at higher magnification. Scale bar: 20 μm. D, Intracellular distribution of YBX1 in rat aortic VSMCs infected with adGFP (adenovirus expressing GFP) or ad-arteridin-FLAG (adenovirus expressing-arteridin-FLAG) was detected by Western blot as indicated. GAPDH is used as a cytoplasmic control; Histone 3 (H3) is used as nucleic control. E, Representative immunofluorescent staining of DAPI (blue), YBX1 (red), and α-SMA (α-smooth muscle-actin; green) in the carotid artery tissue 14 d post balloon injury from the WT and arteridin knock-out (KO) rats. Scale bar: 50 μm. F, Intracellular distribution of YBX1 in carotid artery tissue from the WT and arteridin KO rats subjected to sham or 14 d after balloon injury was detected by Western blot as indicated. GAPDH is used as a cytoplasmic control; H3 is used as nucleic control. G, Relative changes of expression for contractile gene ACTA2 and CNN1 in primary rat VSMCs transfected with lenti-GFP plus scramble small interfering RNA (siRNA), or lenti-arteridin-Remix-FLAG (lenti-Art Remix) with or without co-transfection with siYBX1 (small interfering RNA targeting YBX1), normalized to CYPA (cyclophilin A). Data are median±interquartile range, vs lenti-GFP+scramble, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). H, ChIP-qPCR (chromatin immunoprecipitation quantitative real-time PCR) analysis for YBX1 occupancies at the promoter region of ACTA2 gene in rat VSMCs with or without ad-arteridin-FLAG transfection. Data are median±interquartile range, vs adGFP (adenovirus harboring green fluorescent protein) as indicated, by Mann-Whitney U test (n=4). I, Relative levels of lncPSR following RNA-immunoprecipitation (RIP) using anti-FLAG in rat aortic VSMCs transfected with arteridin-FLAG, normalized to RIP signal using IgG as control. Data are median±interquartile range, vs adGFP, by Mann-Whitney U test (n=4). J, Relative levels of lncPSR following RNA RIP using anti-HA in rat aortic VSMCs transfected with full length or truncated YBX1-HA cDNAs as indicated in E, all signals are normalized to the signal from the corresponding IgG RIP samples. Data are median±interquartile range, vs YBX1-HA as indicated, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). K, Co-IP assay in HEK293 cells cotransfected with rat arteridin-FLAG and rat YBX1-HA with or without RNase A (100 μg/mL) incubation at 37 °C for 20 min before IP using anti-FLAG, followed by Western blot using anti-HA. A indicates adventitia; cyto, cytoplasmic; M, media; and N, neointima; and nuc, nucleic.

Using RNA-immunoprecipitation assay in rat aortic VSMCs expressing arteridin-FLAG or YBX1-HA, we detected a specific interaction of lncPSR with both arteridin (Figure 6I) and YBX1 (Figure 6J). Deletion mutant study showed that lncPSR interacted with YBX1 through its cold shock domain or the C-terminal domain (Figure 6J). RNase A (100 μg/mL) treatment31 before coimmunoprecipitation diminished the Arteiridin-YBX1 interaction in HEK293 cells cotransfected with rat arteridin-FLAG and YBX1-HA (Figure 6K), demonstrating that the formation of this protein complex is RNA dependent, suggesting that lncPSR binding with both arteridin and YBX1 is necessary for arteridin-YBX1 interaction.

Arteridin-YBX1-lncPSR Complex Regulates Transcriptional Reprogramming in VSMCs

Considering the observation that arteridin expression promoted the nuclear translocation of YBX1, we investigated the potential impact of arteridin on the nuclear function of YBX1 by measuring its chromatin occupancy. YBX1 chromatin-binding profiles were mapped in the absence or presence of ectopically expressed arteridin in rat A10 aortic VSMCs. Compared with the empty vector control (NC), arteridin overexpression significantly enhanced the total chromatin occupation of YBX1 (Figure 7A). To also evaluate the nuclear function of lncPSR, we isolated lncPSR-associated chromatin using Chromatin Isolation by RNA Purification (ChIRP) method followed by sequencing (ChIRP-seq) in rat A10 aortic VSMCs. From the ChIRP-seq data, a total of 8294 lncPSR binding sites were identified with ≈4% peaks located to promoter elements, while the remaining peaks were mainly mapped to intergenic or intronic regions (Figure 7B). Remarkably, among the lncPSR binding genes, a total of 3622 genes were shared with YBX1 binding genes, consistent with a model that lncPSR and YBX1 are in the same DNA binding complex for targeted transcription in VSMCs (Figure S7C). By integrating the differentially expressed genes from siPSR (small interfering RNA targeting lncPSR) versus scramble RNA-seq (RNA sequencing) with genes identified by YBX1 chromatin immunoprecipitation–sequencing and lncPSR ChIRP-seq (Figure 7C), 331 genes were identified as potential direct targets of YBX1-lncPSR complex-mediated transcription regulation (Figure 7D and Table S3). Gene Ontology biological process analysis of the 331 genes identified significantly enriched biological processes, including regulation of vascular development, cell proliferation, extracellular matrix organization, cell adhesion, etc (Figure 7E). Among these candidate target genes, some have long been implicated in vascular remodeling, such as ACTA2, KLF5 (Krueppel-like factor 5),32Col1a1 (alpha-1 type I collagen),33Adamts2 (a disintegrin and metalloproteinase with thrombospondin motifs 2),34 and CCL5 (C-C motif chemokine ligand 5) (Figure 7F).26 Using qRT-PCR, we validated that the expression of these candidate target genes was indeed altered in VSMCs with lncPSR or YBX1 knockdown (Figure S7D). Using ChIP-qPCR, we confirmed that YBX1 occupancies were significantly increased in carotid tissue 14 days after balloon injury compared with the sham group, while arteridin KO markedly abolished the injury-induced increase of YBX1 occupancy (Figure 7G). All the evidence supports a critical role of arteridin-YBX1-lncPSR complex in transcriptional regulation during vascular remodeling.

Figure 7.

Figure 7. Arteridin-YBX1 (Y-box binding protein 1)-lncPSR (PSR transcript) complex regulates transcriptome reprogramming in vascular smooth muscle cells (VSMCs). A, Visualization of chromatin occupancy of YBX1 detected by chromatin immunoprecipitation followed by DNA sequencing (chromatin immunoprecipitation–sequencing [ChIP-seq]) in rat A10 thoracic artery VSMCs transfected with arteridin-FLAG plasmid (Arteridin OE‚ green) compared with vector only control (negative control [NC], orange). B, Pie chart depicting the distribution of lncPSR binding sites genome-wide, detected by Chromatin Isolation by RNA Purification followed by DNA sequencing (ChIRP-seq) in untreated rat A10 thoracic artery VSMCs. C, Flow chart depicting strategy of identification of potential lncPSR-arteridin-YBX1 targets. D, Venn diagram depicting overlap among differentially expressed genes (DEGs), YBX1 binding target genes, and lncPSR binding target genes. E, Gene ontology (GO) analysis of the 331 potential target genes of lncPSR-arteridin-YBX1 complex. F, Representative image of YBX1 ChIP-seq (labeled in red) and lncPSR ChIRP-seq (labeled in blue) at target gene loci. Visualized using Integrated Genome Browser. Rsp18 gene is shown as an NC. G, ChIP-qPCR (chromatin immunoprecipitation quantitative real-time PCR) analysis for YBX1 occupancies at specific promotor regions as indicated in the carotid artery tissue from the wild-type (WT) and arteridin knock-out (KO) rats subjected to sham or 14 d after balloon injury. Data are median±interquartile range, vs sham as indicated, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). H, Endogenous lncPSR expression detected by qRT-PCR (quantitative real-time PCR) in rat aortic VSMCs transfected with adGFP with scramble small interfering RNA (siRNA), ad-arteridin-FLAG with scramble siRNA, or ad-arteridin-FLAG with siYBX1 (small interfering RNA targeting YBX1) as indicated. Signals are normalized to CYPA (cyclophilin A). Data are median±interquartile range, vs adGFP+scramble, by Kruskal-Wallis test with Dunn post hoc analysis (n=4). I, Representative image of YBX1 ChIP-seq and lncPSR ChIRP-seq binding peaks on phenotype switching regulator (PSR) gene locus, visualized using Integrated Genome Browser. J, Transcriptional activity of PSR promoter in rat aortic VSMCs cotransfected with pcDNA5 or Arterdin-FLAG vector. Data are median±interquartile range, vs pcDNA5, by Mann-Whitney U test (n=5). K, Interaction between rat arteridin and the promoter region of rat PSR gene detected by Electrophoretic mobility shift assay. Chr indicates chromosome; and pGL3‚ plasmid gene luciferase 3.

Interestingly, we noticed that PSR (LOC680254) gene was one of the candidate downstream targets of arteridin-YBX1-lncPSR complex based on the integrated analysis described above (Table S3). Indeed, the PSR gene was significantly induced by arteridin overexpression but abolished by YBX1 knockdown in rat aortic VSMCs (Figure 7H), indicating a YBX1-dependent positive auto-regulatory loop. In rat A10 aortic VSMCs, the lncPSR interacting regions detected by ChIRP-seq overlapped with a YBX1 binding motif (Figure S7E) identified in the promoter region of PSR gene (Figure 7I). These data show that YBX1 is a potential direct transcriptional factor for PSR transcription. Moreover, using dual-luciferase reporter assay, we found that arteridin expression significantly induced the transcriptional activity of the PSR promoter (Figure 7J). However, electrophoretic mobility shift assay failed to detect a direct binding of arteridin protein to the promoter sequence of PSR (Figure 7K), suggesting that arteridin may function as a co-transcription factor. Together, using PSR gene as an example, we showed that arteridin-YBX1-lncPSR complex regulates transcriptional activity of its target genes, with YBX1 as the direct DNA binding factor while arteridin is the cofactor of YBX1.

VSMC-Specific lncPSR Knockdown Attenuated Ang II–Induced Vascular Remodeling.

Considering that lncPSR was identified to be induced during hypertensive vascular remodeling and was necessary for VSMC phenotype switching, we explored whether lncPSR could serve as a therapeutic target in a preclinical vascular remodeling model by Ang II treatment in mice.35,36 Using an SM22a promoter-driven AAV2 vector37 injected through the tail vein,37 we expressed an shRNA sequence targeting mouse lncPSR (AAV-shPSR) or a scrambled shRNA (AAV-scramble) with eGFP tag specifically in VSMC in vivo. One week after injection, the mice were chronically treated with Ang II minipump (1000 ng/kg/min) for one month,36,38 and monitored with weekly tail-cuff blood pressure measurement (Figure 8A). The expression level of lncPSR was significantly downregulated by AAV-shPSR as expected (Figure 8B). Immunofluorescent costaining of eGFP and α-SMA validated the VSMC-specific expression pattern of the SM22a promoter-driven AAV2 vector in the aortic tissue from mice transfected with AAV-scramble for two weeks (Figure S8A). Immunofluorescent costaining of arteridin and α-SMA demonstrated that lncPSR was effectively knocked down in the VSMCs of the aorta (Figure S8B). LncPSR knockdown did not influence the blood pressure at baseline, but after 1-month of Ang II treatment, the AAV-shRNA injected mice had lower systolic blood pressures by about 20 to 30 mm Hg than the AAV-scramble-injected mice (Figure 8C). Ang II infusion induced marked vascular remodeling in the AAV-scramble-injected mice as evidenced by increased media layer thickness, media to lumen area ratio, and collagen deposition of thoracic aortae (Figure 8D–8G). However, these pathological features were reversed by AAV-shPSR treatment (Figure 8D through 8G), accompanied by attenuated VSMC phenotype switching, as demonstrated by VSMC contractile protein levels (Figure 8H). These results demonstrated that AAV-mediated lncPSR knockdown ameliorated Ang II–induced vascular remodeling and VSMC phenotypic switching.

Figure 8.

Figure 8. Vascular smooth muscle cell (VSMC)–specific lncPSR (PSR transcript) knockdown attenuated Ang II (angiotensin II)–induced hypertension and vascular remodeling in mice. A, Schematic illustration of the study design. B, Expression of lncPSR detected by qRT-PCR (quantitative real-time PCR) in the aortic tissue of mice injected with SM22a (smooth muscle protein 22a) promoter-driven miR-15 (microRNA 15)-scaffolded adeno-associated virus stereotype 2 (AAV2) harboring scramble short hairpin RNA (shRNA; scramble) or shRNA targeting mouse lncPSR (shPSR), followed by infusion of saline or Ang II (1000 ng/kg/min) by minipump for 28 d. The relative expression levels are normalized to GAPDH. Data are mean±SEM, vs scramble+saline or scramble+Ang II as indicated, by 1-way ANOVA with Bonferroni post hoc analysis (n=6). C, Systolic blood pressure (SBP) measured by tail-cuff method in mice injected with AAV2 scramble shRNA or AAV-shPSR every 7 d after Ang II or saline infusion. Data are mean±SEM, vs scramble+Ang II, by repeated measures 2-way ANOVA with the Geisser-Greenhouse correction followed by Sidak post hoc analysis (n=8). D, Representative histological sections of thoracic aorta from mice injected with AAV2 scramble shRNA or AAV-shPSR with Ang II or saline infusion for 28 d, stained with hematoxylin and eosin (H&E; morphology) and Masson trichrome staining (fibrosis). Scale bar: 100 μm. E, Quantification of media thickness of thoracic aorta in mice injected with AAV2 scramble shRNA or AAV-shPSR with Ang II or saline infusion for 28 d. Data are mean±SEM, vs scramble+saline or scramble+Ang II as indicated, by 1-way ANOVA with Bonferroni post hoc analysis (n=8 or 9 mice/group). F, Quantification of media-to-lumen ratio of thoracic aorta in mice injected with AAV2 scramble shRNA or AAV-shPSR with Ang II or saline infusion for 28 d. Data are mean±SEM, vs scramble+saline or scramble+Ang II as indicated, by 1-way ANOVA with Bonferroni post hoc analysis (n=8 or 9 mice/group). G, Quantification of fibrosis volume of thoracic aorta in mice injected with AAV2 scramble shRNA or AAV-shPSR with Ang II or saline infusion for 28 d. Data are mean±SEM, vs scramble+saline or scramble+Ang II as indicated, by 1-way ANOVA with Bonferroni post hoc analysis (n=8 or 9 mice/group). H, Representative (top) and quantification (bottom) of Western blot for protein levels of VSMCs contractile marker genes α-SMA (α-smooth muscle-actin), Calponin, and SM22 in thoracic aorta in mice injected with AAV2 scramble shRNA or AAV-shPSR with Ang II or saline infusion for 28 d. The data were normalized to GAPDH. Data are mean±SEM, vs scramble+saline or scramble+Ang II as indicated, by 2-way ANOVA with Sidak post hoc analysis (n=6). I, Schematic illustration for the function and mechanism of lncPSR and arteridin protein in VSMCs phenotype switching and vascular remodeling. YBX1 indicates Y-box binding protein 1.

Discussion

Here, we reported the identification and functional characterization of a previously uncharacterized VSMC-abundant lncRNA, named PSR, in the regulation of VSMC phenotypic reprogramming and vascular remodeling. Genetic KO of PSR gene in rats resulted in reduced neointima formation after carotid artery balloon injury associated with blunted VSMC phenotype switching and proliferation. Moreover, VSMC-specific knockdown of lncPSR using AAV-shRNA attenuated Ang II–induced hypertensive vascular remodeling, indicating that it may serve as a potential therapeutic target to treat vascular remodeling under diseased conditions.

More interestingly, although the PSR transcript was initially annotated as a long noncoding RNA, we found that it also encoded a protein, named arteridin. This was demonstrated by direct detection of the endogenous protein in aortic tissues using a custom-made arteridin-specific antibody, in cells transfected with the cDNA construct and in a public dataset of rat and human proteome. By establishing an arteridin ablation rat without disrupting the transcription of lncPSR, we found that arteridin ablation phenocopied the PSR-KO in carotid artery balloon injury-induced neointima formation, VSMC phenotype switching, and proliferation, suggesting that arteridin is the essential functional entity of lncPSR.

Integrated and unbiased approaches, including Ribo-seq (ribosome sequencing), RNA-seq, and mass spectrometry have uncovered that multiple annotated lncRNAs possess protein/peptide coding potential through canonical or noncanonical open reading frames, and many of these protein/peptide products have demonstrated essential biological roles.39 For example, Myoregulin,18 DOWRF (dwarf open reading frame),40 and several other muscle-specific micro-peptides 41 are products of previously annotated lncRNAs and show an important role in regulating muscle performance by modulating calcium homeostasis. Other micro-peptides are also identified in mitochondrial regulation of fatty acid metabolism and respiratory activities.42,43 While most of the studies on these newly reported peptides focus on the protein component, the potential function of the hosting RNA transcripts, independent of their protein product is rarely explored. For example, a peptide SPAR (small regulatory polypeptide of amino acid response) was first reported in muscle regeneration,44 but later, the noncoding transcript itself was also found to enhance the myogenic effect from the SPAR peptide,45 raising the possibility that both coding and noncoding products from a single gene may be functional. In most reported cases, however, the encoded peptides are either the sole functional products18,40 or operate independently from the noncoding RNA transcripts in terms of outcome and mechanisms.46 Unexpectedly, using a series of expression constructs, including a full-length lncPSR cDNA, a noncoding lncPSR full-length mutant, and an artificial open reading frame with altered arteridin codon utilization in VSMCs, we demonstrated that both the noncoding lncPSR transcript and the peptide product arteridin showed similar functional outcome in promoting phenotypic reprogramming in VSMCs. However, in VSMCs isolated from arteridin KO rats, lncPSR transcript overexpression failed to induce phenotype switching, suggesting that both lncPSR transcript and arteridin protein are required and are involved in the same regulatory circuit and cellular process in driving VSMC phenotype switching.

At the molecular level, we found both arteridin and lncPSR are part of the same molecular complex with a transcription factor YBX1. Based on direct immunofluorescent imaging and cellular fractionation, we found that the intracellular localization of YBX1 is modulated by arteridin. RNA-seq, YBX1 chromatin immunoprecipitation–sequencing, and lncPSR ChIRP-seq analyses further demonstrated that arteridin and lncPSR confer YBX1 targeting to a specific subset of genes known to be important for VSMC phenotype reprogramming under pathological stimulation (Figure 8I). YBX1 is both a transcription factor and an RNA binding protein, which was thoroughly studied in the regulation of tumor growth and metastasis.47,48 It has been reported that YBX1 plays an essential role in neointima formation through regulating CCL5 gene transcription in VSMCs,26 and YBX1 is also a known repressor of VSMC contractile protein gene ACTA2.27,29,30,49 In this study, we further showed that YBX1 is a phenotype switching inducer, which was both necessary and sufficient to induce VSMCs phenotype switching. YBX1 was identified as an arteridin interacting protein through arteridin IP followed by LC-MS (liquid chromatography–mass spectrometry). Using in vivo and in vitro studies, we demonstrated that arteridin-mediated VSMC phenotype switching is YBX1-dependent. The potential downstream targets of arteridin-YBX1-lncPSR complex, including known vascular homeostasis regulators KLF5,32 CCL5,26 Adamts2,34 etc, are significantly correlated with biological processes, including vascular development, cell proliferation, cell migration, and extracellular matrix organization, etc, which are important in vascular remodeling. These support that this newly identified arteridin-YBX1-lncPSR complex regulates vascular remodeling through transcriptional regulation in VSMCs. Although we did not discover the direct interaction between other classic VSMC phenotype switching regulators, such as Myocardin, Serum Response Factor (SRF), or KLF4 (krueppel-like factor 4)50-52 in our LC-MS data, while these factors are not among the direct downstream targets of arteridin-YBX1-lncPSR transcriptional regulation complex either, the relationship between arteridin and these factors are still worth studying in the future. For example, YBX1 has been reported to regulate KLF4 transcript stability by serving as RNA binding protein,53,54 suggesting the possibility that arteridin and YBX1 may exert their function through targeting RNA.

Beyond their coordinated impact on YBX1 localization and chromatin targeting, we found that arteridin is also a potent activator to its host gene PSR transcription by serving as a cofactor of YBX1. Although the functional significance of this auto-regulatory scheme remains to be fully demonstrated, it adds a potential mechanism that amplifies stress signaling to yield a robust gene reprogramming in VSMC to initiate or maintain phenotypic switching at molecular and cellular levels.

Compared with mRNAs, lncRNAs have lower expression levels and sequence conservation, yet retain critical evolutionary conserved functions.55 The lncPSR transcript and arteridin protein have high sequence conservation between rat and mouse, but has relative low sequence conservation with human, though the genomic locus relationship of PSR gene is conserved among these 3 species. In this study, the functional conservation of lncPSR and arteridin was illustrated as follows: (1) The expression of lncPSR was upregulated in rat hypertensive vascular remodeling model, rat carotid artery balloon injury model, mouse Ang II–induced hypertensive vascular remodeling model, and human mesenteric arteries with neointima formation. (2) Immunohistochemistry staining showed that human arteridin is enriched in media layer of mesenteric arteries, with an increased expression in the VSMCs in the neointima, which is consistent with that in rat. (3) In both rat and human aortic VSMCs, knockdown of lncPSR maintained the contractile phenotype. (4) The interaction between arteridin and YBX1 is conserved in rat and human.

To expand the translational implication of our findings, and considering AAV is a promising candidate for gene therapy because of its relative high transfection efficiency and mild immune response,56 we performed a preclinical trial using AAV to treat Ang II–induced hypertensive vascular remodeling in mice. VSMC-specific knockdown of lncPSR by SM22a promoter-driven miR-15 (microRNA 15)-scaffolded AAV2 attenuated the Ang II–induced hypertension and vascular remodeling, shedding light on the translational potential of lncPSR to be a therapeutic target for the treatment of VSMC phenotype switching–related vascular remodeling.

Finally, considering the prevalence of coding potential among the previously annotated lncRNAs in mammalian genome, our findings highlight the possible involvement of both noncoding and coding products from a single gene with same or opposite cellular function. Therefore, mechanistic interpretation of gene function may require additional consideration and rigorous evaluation of both coding and noncoding products.

Article Information

Acknowledgments

The authors would like to thank Prof. Runsheng Chen and Prof. Hong Zhang from Institute of Biophysics, Chinese Academy of Sciences for commenting on this manuscript. The authors would like to thank Dr. Jiahuan Chen, Zilong Geng, and Yingmei Lou from the Dr Bing Zhang’ s lab at Shanghai Jiao Tong University for technical assistance. The authors would like to thank Professor Yazhou Wu from Department of Health Statistics, the Third Military Medical University, and Professor Li Zhou from Department of Epidemiology, Chongqing Medical University for statistical methodological consulting. The authors would like to thank Professor Pedro A. Jose from Division of Renal Diseases & Hypertension, The George Washington University School of Medicine & Health Sciences for language polishing. The authors also thank Dr. Gang Wu from Institute of Medicine and Equipment for High-Altitude Region, College of High-Altitude Military Medicine, The Third Military Medical University, for the gifting of cDNA generated from rat pulmonary arterial endothelial cells.

Supplemental Materials

Supplemental Methods

Figures S1–S8

Tables S1–S4

References 57–67

Supplemental File of Uncropped Western blots

Major Resources Table

Nonstandard Abbreviations and Acronyms

α-SMA

smooth muscle α-actin

AAV

adeno-associated virus

ACTA2

actin alpha 2, smooth muscle

Ang II

angiotensin II

ChIRP-seq

chromatin isolation by RNA purification–sequencing

KO

knock-out

lncRNA

long noncoding RNA

ORF

open reading frame

PDGF

platelet-derived growth factor

PSR

Phenotype Switching Regulator

SHR

spontaneously hypertensive rat

shRNA

short hairpin RNA

SM22

smooth muscle protein 22

VSMC

vascular smooth muscle cells

YBX1

Y-box binding protein 1

Disclosures None.

Footnotes

*J. Yu, W. Wang, J. Yang, and Y. Zhang contributed equally.

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.122.321080.

For Sources of Funding and Disclosures, see page 785.

Correspondence to: Chunyu Zeng, MD, PhD, Department of Cardiology, Daping Hospital 10th Changjiang Rd, Yuzhong District, Chongqing, China 400042, Email
Correspondence to: Gengze Wu, MD, PhD, Department of Cardiology, Daping Hospital 10th Changjiang Rd, Yuzhong District, Chongqing, China 400042, Email
Correspondence to: Yibin Wang, PhD, Signature Program in Cardiovascular and Metabolic Diseases Duke-NUS School of Medicine, Singapore 169857, Email

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