Introduction

Editorial, see p 1863

Calcific aortic valve disease (CAVD) is the most common heart valve disease. It is a chronic disorder in which ectopic mineralization of the aortic valve leaflets plays a major role.^1,2 Studies have highlighted that age, dyslipidemia, diabetes mellitus, hypertension, and a bicuspid aortic valve are risk factors for CAVD.^3–5 Transcriptomic and expression quantitative trait loci mapping studies indicate that genes related to osteogenesis are overrepresented in surgically explanted human CAVD.^6,7 The expression of several key regulators of the mineralization process is increased in CAVD,^8,9 including runt-related transcription factor 2 (RUNX2), a master transcription factor that determines the osteogenic fate of cells.¹⁰RUNX2 is a potential important driver of the mineralization in the aortic valve.^7,11 Studies also underscored that an upstream regulator of RUNX2 such as NOTCH1 may exert an important control over the osteogenic fate of valve interstitial cells (VICs), the main cellular component of the aortic valve.¹² Frameshift mutations and gene variants of NOTCH1 have been described in patients with mineralized bicuspid aortic valve (a valve with 2 leaflets instead of 3) and tricuspid aortic valves.^13,14 The molecular processes that control the expression of osteogenic genes, including NOTCH1, are incompletely understood.

Growing evidence suggests that long noncoding RNAs (lncRNAs) are important key regulators that fine-tune gene expression and cell differentiation in response to different stimuli.¹⁵ LncRNAs exert a control on cell responses by a variety of mechanisms. LncRNAs have been shown to interact with proteins, to interfere with microRNAs by acting as molecular sponges, to modify the epigenome, and to interfere with gene expression by being recruited to gene promoters.¹⁶ In addition, some lncRNAs are involved in the control of gene imprinting and are imprinted, showing parent-of-origin allelic expression.^17,18 Gene imprinting is regulated by DNA methylation. Imprinted genes, including the lncRNA H19 (H19)–insulin like growth factor 2 (IGF2) gene locus, contain differentially methylated regions that control parent-of-origin expression.¹⁹ The estimated large number of lncRNAs and the varied mechanisms by which they regulate gene expression suggest that they may play complex roles in different disorders, including CAVD. Whether epigenetic-related dysregulation of lncRNA occurs during CAVD is currently unknown. In this work, we discovered that DNA hypomethylation in the promoter region of H19 during CAVD leads to an overexpression of this lncRNA, which, in turn, promotes the osteogenic fate of VICs by fine-tuning the expression of NOTCH1.

Methods

Expanded materials and methods sections are available in the Data Supplement.

Procurement of Tissues for Analyses

We examined stenotic aortic valves (CAVD) that were explanted from patients at the time of aortic valve replacement. Control noncalcified aortic valves were obtained during heart transplantation procedures. Patients with a history of rheumatic disease, endocarditis, and inflammatory diseases were excluded. Valves with moderate to severe aortic valve regurgitation (greater than grade 2) were excluded. The protocol was approved by the local ethics committee, and informed consent was obtained from the subjects.

VIC Isolation

Human VICs were isolated from control nonmineralized aortic valves obtained from patients undergoing heart transplantation. VICs were isolated, and the purity of the cell preparation was confirmed as previously described.²⁰

Real-Time Polymerase Chain Reaction

RNA was extracted from valves explanted from patients and from cells during in vitro experiments. Total RNA was isolated with RNeasy micro kit from Qiagen (Toronto, ON, Canada). The RNA extraction protocol was performed according to manufacturer’s instructions. RNA 1 μg was reverse transcribed with the QuantiTec Reverse Transcription Kit from Qiagen. Quantitative real-time polymerase chain reaction (qPCR) was performed with the QuantiTec SYBR Green PCR kit from Qiagen on the Rotor-Gene 6000 system (Corbett Robotics Inc, San Francisco, CA).

In Situ Hybridization on Cells

The in situ hybridization procedure was performed according to a protocol prepared by R.W. Dirks at the Leiden University Medical Center (the Netherlands) and posted on the Exiqon Web site. Slides were mounted and analyzed with an UltraVIEW spinning disk confocal imaging system (objective 60× oil, 1.4 NA, PerkinElmer Life and Analytic Sciences, Waltham, MA) equipped with a cooled electron multiplying charge-coupled device camera at −50°C (Hamamatsu Photonics K.K., Hamamatsu-shi, Japan) and driven by Volocity software, version 6.0.1 (PerkinElmer Life and Analytic Sciences). Image processing was performed with ImageJ version 1.49 (National Institutes of Health, Bethesda, MD).

In Situ Hybridization on Tissues

The in situ hybridization procedure for frozen sections was performed according to a protocol prepared by Dr Aurora Esquela-Kerscher at Yale University and posted on the Exiqon Web site. Slides were mounted, and confocal images were acquired with a Zeiss microscope LSM800 driven by the Zen software (objective 63× oil, 1.4 NA, Zeiss, North York, ON, Canada). Image processing was performed with ImageJ version 1.49 (National Institutes of Health).

RNA Sequencing

Gene expression was evaluated by the Illumina HiSeq 2000 platform. TopHat and Cufflinks were used to align and assemble reads. Gene expression levels between groups of valves were compared with Cuffdiff, DESeq, and edgeR as previously described.⁷ Correction for multiple testing was performed with Benjamini and Hochberg correction. LncRNAs with an adjusted value of P<0.05 in at least 1 software program (Cuffdiff, DESeq, edgeR) were considered differentially regulated.

Whole-Genome DNA Methylation

Methylation sites were interrogated on the Illumina Infinium HumanMethylation450 BeadChip testing 485 513 CpG sites located in the gene promoter, 5′ untranslated region, first exon, gene body, and 3′ untranslated region, densely methylated regions, and methylation islands.

Genotyping rs2839704 in Genomic DNA and cDNA

Genomic DNA (gDNA) from 32 patients with tricuspid calcified aortic valve and 33 patients with tricuspid control nonmineralized aortic valve (obtained from heart transplantations) were genotyped with the Illumina HumanOmni2.5-8 BeadChip. From these samples, 28 were identified as heterozygotes for rs2839704. The aortic valve tissues of these subjects were then used for RNA extraction, followed by cDNA synthesis, PCR amplification, and Sanger sequencing to genotype rs2839704.

Pyrosequencing

DNA was purified from aortic valve samples with the QIAamp DNA mini kit (Qiagen). H19 DNA methylation levels were measured with bisulfite pyrosequencing, which combines sodium bisulfite conversion of gDNA (500 ng; EpiTech Bisulfite Kit, Qiagen), PCR amplification of bisulfite-converted DNA, and sequencing of the amplicons on a PyroMark Q24 (Qiagen).

Transfections

HEK-293T cells were seeded on poly-l-lysine (Sigma-Aldrich, Oakville, ON, Canada) and transfected with the calcium phosphate technique. VICs, Saos2, and COS7 were transfected with the nanojuice transfection reagent (EMD Millipore, VWR, Montreal, QC, Canada). For siRNA treatment, cells were transfected by use of HiPerfect reagent (Qiagen) with 300 to 600 ng siRNA (Qiagen) according to the manufacturer’s instructions.

ELISA

Bone morphogenetic protein 2 (BMP2) and p53 were measured by ELISA (R&D Systems, Cedarlane, ON, Canada) according to manufacturer’s instructions and normalized for protein content.

Western Blotting

Cell extracts were boiled for 5 minutes, and proteins were loaded onto polyacrylamide gels followed by electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked with TBS-Tween containing 5% BSA (Sigma-Aldrich) and incubated with RUNX2, p53, NOTCH1 (Cell Signaling Technology, Danvers, MA), CALNEXIN (Santa Cruz Biotechnologies, Dallas, TX), or β-Actin (Sigma-Aldrich) antibodies overnight at 4°C. Membranes were then washed and incubated with horseradish peroxidase–labeled secondary antibodies (Cell Signaling Technology). Detection was done with Clarity Western ECL Substrate (BioRad, Mississauga, ON, Canada).

Determination of Calcium Concentrations

Calcium content in cell cultures or valves was determined by the Arsenazo III method (Synermed, Monterey Park, CA). Results were normalized to protein contents for cell culture experiments or gram of wet weight for valve tissues.

Alizarin Red Staining of Cultured Cells

Cells were stained with 2% Alizarin Red solution. Alizarin Red solution was prepared by dissolving 2 g Alizarin Red (Sigma-Aldrich) in 100 mL distilled water and mixing well, and the pH was adjusted to 4.2 with 10% NH₄OH.

Luciferase Assay

HEK-293T or Saos2 cells were transfected with vectors encoding for NOTCH1 promoter or RBPJ response elements (×4) fused to the firefly luciferase gene, together with a vector encoding for renilla luciferase as a reporter for transfection efficiency. Luciferase activities were measured at 48 hours after transfection with the Promega Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI) according to the manufacturer’s protocol.

H19 Pull-Down

HEK-293T cells were transfected with H19-S1 or empty pCDNA3.1. Cells were cross-linked with 10 mL of 0.37% formaldehyde in PBS1X. Cell lysates were centrifuged at 13 000 rpm at 4°C for 10 minutes and precleared with 40 µL avidin agarose beads (ThermoScientific, ON, Canada) for 1 hour at 4°C on a rotator. The cleared lysates were mixed with 40 µL of streptavidin beads (ThermoScientific) and incubated on a rotator at 4°C for 3 hours.

Chromatin Immunoprecipitation

HEK-293T cells were transfected with H19 or empty pCDNA3.1. Cells were crosslinked with 0.2 mL of a 10% formaldehyde solution (Sigma-Aldrich). Cells were then centrifuged at 1400g for 5 minutes at 4°C. Pellets were resuspended in PBS1X containing protease inhibitor cocktail and centrifuged at 1400g for 5 minutes at 4°C. Pellets were then resuspended in lysis buffer and rocked at room temperature for 10 minutes. After centrifugation, pellets were resuspended in low-salt buffer and sonicated to shear the DNA to an average length of 100 to 400 base pairs (bps). P53 antibodies or normal mouse serum (Cell Signaling Technology) were incubated with proteins G Dynabeads (Life Technologies) for 6 hours before DNA samples were added to the antibodies/Dynabeads mixtures and incubated at 4°C overnight. The next day, the samples were washed first with low-salt buffer, then with high-salt buffer, and finally with Tris-EDTA buffer. The complex was then eluted from Dynabeads by adding 210 µL elution buffer at 65°C for 1 hour. The eluted samples were reverse cross-linked by incubation at 65°C overnight. DNA fragments were purified with phenol chloroform, and samples were analyzed by qPCR.

Statistical Analyses

Continuous data were expressed as mean±SEM and compared by use of the Student t test (when 2 groups were compared) or 1-way ANOVA to test the effect of group (when >2 groups were compared). Post hoc Tukey analyses were done when the P value of the ANOVA was <0.05. Categorical data were expressed as percentage and compared with the Fisher exact test. Age-adjusted P value for the expression of H19 in valve tissues was calculated with multiple logistic regression analysis. A value of P<0.05 was considered significant. Statistical analysis was performed with a commercially available software package JMP 10.0 or Prism 6.0.

Results

H19 Is Highly Expressed in Calcified Aortic Valve and Associated With Indexes of Disease Activity

We investigated the expression of annotated and functional lncRNAs recovered from the lncRNA database (lncrnadb version 2.0). We performed RNA sequencing on 9 tricuspid CAVD and 10 control nonmineralized aortic valves (Table 1). LncRNAs differentially expressed between the 2 groups are presented in Figure 1A. Compared with control nonmineralized aortic valves, we found that 4 lncRNAs were upregulated and 5 were significantly downregulated in CAVD. Among the 4 upregulated lncRNAs in CAVD, H19 attracted our interest because this noncoding gene is imprinted, showing parent-of-origin epigenetic signatures, and after birth its expression is restricted to the skeletal muscle and the heart.²¹ The expression and role of H19 in the aortic valve have not been documented previously. We confirmed the finding of the RNA sequencing experiment by using qPCR in independent and larger groups of control (n=44) and CAVD (n=106) valves (Table 2). H19 was increased by 7.5-fold in mineralized, stenotic valves compared with control nonmineralized aortic valves (age-adjusted P<0.0001; Figure 1B). In 6 sclerotic and thickened aortic valves, which represent the early stage of CAVD, we measured the level of H19 to evaluate whether the upregulation of H19 is an early event. Compared with control nonmineralized aortic valves, the expression of H19 was increased by 3.3-fold in sclerotic valves (Figure 1C), suggesting that H19 is upregulated early during CAVD. A conserved microRNA (miR675) can be produced from the exon1 of H19. However, after qPCR, we could not detect miR-675 in control and mineralized aortic valves. RNA fluorescent in situ hybridization and confocal microscope imaging in mineralized aortic valves indicated that H19 was expressed with a predominant nuclear or perinuclear localization (Figure 1D). Using fluorescent in situ hybridization and immunofluorescence, we found that H19 was coexpressed with cells positive for vimentin, a marker of VICs (Figure 1E). We next isolated human VICs from nonmineralized valves, and we confirmed by fluorescent in situ hybridization and confocal microscope imaging that H19 was located in the nucleus and in the cytosol (Figure 1F). Quantitative assessment showed that ≈65% of isolated VICs were positive for H19 in fluorescent in situ hybridization (Figure 1F). Using qPCR, we found that although H19 is not expressed in several cell lines, including 293T, COS7, and Saos2, it is expressed in human primary VICs (Figure 1G). In mineralized aortic valves, we next found that the amount of calcium was significantly elevated in tissues with a higher level of H19 (≥1.2 copies per GAPDH, median; Figure 1H). In 36 patients for whom we had 2 preoperative echocardiographic analyses separated by at least 6 months, we calculated the annualized progression rate by using the peak gradient. In patients with a faster progression rate of aortic stenosis (≥8 mm Hg/y, median), the level of H19 was increased by 2.9-fold (Figure 1I). These data thus indicate that H19 is expressed by VICs and that its level in mineralized aortic valves is related to indexes of disease activity.

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DNA Methylation Is Associated With the Expression of H19

H19 is paternally imprinted, meaning that it is expressed from only the maternal allele. Dysregulation of the differentially methylated region in the imprinting control region (ICR) has been associated with a loss of imprinting in different pathological conditions. Using patients heterozygous for the H19 polymorphism rs2839704 (exon 5), we assessed allele-specific expression of H19 (Figure 2A).²² cDNA (synthesized from mRNA) and gDNA were isolated from aortic valves of control (n=12) and CAVD (n=16) tissues, which showed heterozygosity in gDNA for rs2839704. Although gDNA showed heterozygosity for rs2839704 (AG), cDNA showed a homozygous expression pattern for all the valve tissues (AA or GG), indicating monoallelic expression of H19 in both control and CAVD (Figure 2B). These data suggested that overexpression of H19 in CAVD was not associated with a loss of imprinting. To capture the potential CpG site(s) regulating H19 gene expression, we profiled aortic valves for gene expression and DNA methylation. In 21 CAVD tissues, whole-genome gene expression profile and genome-wide DNA methylation marks were obtained. Results were analyzed considering a region spanning the H19 gene on chromosome 11 and containing 45 putative methylated regions (Figure 2C). DNA methylation level in 6 different sites located in the differentially methylated region and ICR was not associated with the expression level of H19 (Figure 2C). However, DNA methylation in exon 1 (cg15963714) and DNA methylation in the promoter region (−388 bp from the transcription start site; cg25437674) were both significantly associated with H19 levels (Figure 2C). Valvular expression of H19 was inversely related to the CpG methylation (cg25437674) level in the promoter region (r²=0.373, P=0.002; Figure 2D). These results were confirmed with bisulfite pyrosequencing in 58 aortic valves (Table 3). The level of CpG methylation in the promoter region of H19 was significantly reduced in CAVD tissues compared with control nonmineralized aortic valves (Δ=−2.3%; P<0.0001; Figure 2E). In 53 valves, there was enough tissue to measure both the DNA methylation (by pyrosequencing) and H19 levels. In valves with lower promoter DNA methylation (<46.8%, median), the expression of H19 was increased by 4.0-fold (Figure 2F). We next treated VIC cultures with 5-aza-2’-deoxycytidine, a DNA demethylating agent, for 24 hours and measured the level of H19 RNA. Treatment with 5-aza-2’-deoxycytidine increased the level of H19 by 2.5-fold (Figure 2G). Taken together, these data suggest that the hypomethylation of CpG in the promoter region of H19 during CAVD is associated with the expression of its lncRNA.

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H19 Promotes the Osteogenic Transition of VICs

Considering that VICs express H19 and that its level is increased during CAVD, we next hypothesized that H19 may contribute to reprogram VICs toward an osteogenic phenotype. VIC cultures were treated with a mineralizing medium for 7 days, and osteogenic genes were measured after a silencing of H19. Silencing H19 in VICs (Figure 3A) negated the increase in osteogenic genes: osteocalcin (BGLAP), BMP2, and RUNX2 (Figure 3B–3D). Conversely, the transfection of VICs with a vector encoding for H19 increased the level of transcripts encoding for BMP2 and RUNX2 (Figure 3E and 3F). We next measured BMP2 at the protein level using an ELISA. Silencing H19 negated the osteogenic medium–induced increase in BMP2 in VICs (Figure 3G). In the fibroblast cell line COS7, which does not express H19 (Figure 1G), a transfection with a vector encoding for H19 induced an important increase in RUNX2 at the protein level, confirming that H19 induces a strong osteogenic phenotype (Figure 3H). In VICs, after treatment with the osteogenic medium for 7 days, the expression of cadherin 11 (CDH11), a marker of dystrophic mineralization in VIC cultures,²³ was not modified (Figure I in the online-only Data Supplement). However, a knockdown of H19 reduced by 30% the level of mRNA encoding for CDH11 (Figure I in the online-only Data Supplement). On the other hand, the expression of α-smooth muscle actin (ACTA2) was reduced by the osteogenic medium, whereas silencing H19 did not modify its expression (Figure II in the online-only Data Supplement). We next documented the role of H19 in the mineralization of VICs. VIC cultures were treated for 7 days with the mineralizing medium, and the levels of calcium were determined with Arsenazo III, which is specific for calcium and does not cross-react with other divalent ions. After 7 days of treatment with the mineralizing medium, the amount of calcium increased by 93% in VIC cultures (Figure 3I). A knockdown of H19 reduced the mineralization of VICs by 65% (Figure 3I), whereas a transfection of a vector encoding for H19 increased the mineralization process by 141% (Figure 3J). The promineralizing effect of H19 was next corroborated with Alizarin Red staining of VIC cultures. In this experiment over 7 days, we found that transfection of H19 increased the mineralization of VIC cultures, whereas treatment with noggin, an inhibitor of BMP, significantly reduced this process (Figure 3K).

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H19 Is a Negative Regulator of NOTCH1

Cell fate and osteogenic transition in VICs are, at least in part, under the control of the NOTCH1 pathway and its downstream target HEY1, a repressor of BMP2 and RUNX2. Impaired NOTCH1 signaling has been shown to promote an osteogenic program in VICs.²⁴ We thus measured the mRNA encoding for NOTCH1and HEY1 in VICs after transfection of H19. After 7 days, we found that the overexpression of H19 in isolated VICs led to a lower expression of NOTCH1 and HEY1 mRNAs (Figure 4A and B). Conversely, a knockdown of H19 led after 48 hours to an increase in transcripts encoding for NOTCH1 (Figure 4C). After activation of NOTCH1, the notch intracellular domain (NICD) is cleaved by the gamma secretase complex and then associates with a complex of coactivator proteins, including recombining binding protein suppressor of hairless (RBPJ/CBF1), which acts as a transcription factor in the nucleus. We next measured by Western blotting the level of NICD in isolated VICs. Silencing H19 in VICs increased significantly the level of NICD (Figure 4D). To determine the impact of H19 on downstream signaling, we next measured the response of a promoter containing RBPJ-responsive elements in a luciferase assay. We found that 293T cells, which do not express H19 (Figure 1G), were transfected with pGL4 vector containing the firefly luciferase and RBPJ-responsive elements. Cells were also cotransfected with a vector encoding for H19 or a control scrambled sequence. Forty-eight hours after transfection, we found in unstimulated cells that H19 reduced by 48% the activity of the promoter containing RBPJ-responsive elements (Figure 4E). These results are thus in line with the response observed in VICs, in which a knockdown of H19 resulted in higher levels of NICD. We next performed a rescue experiment by transfecting VIC cultures with a vector encoding for the NICD, and we measured the level of mineral in response to transfection with H19. We found that cotransfection with a vector encoding for NICD rescued cell cultures and significantly reduced H19-induced mineralization of VIC cultures after 7 days of treatment with the mineralizing medium (Figure 4F).

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H19 Prevents the Binding of p53 to the Promoter of NOTCH1

Considering that H19 affected the levels of NOTCH1, we next hypothesized that H19 may affect the promoter region of NOTCH1. Luciferase promoter assay performed in 293T cells showed that a transfection of a vector encoding for H19 reduced by 43% the promoter activity of NOTCH1 (Figure 5A). The transcription factor p53 (TP53) is a positive regulator of NOTCH1. In 293T cells, transfection of a vector encoding for p53 increased the NOTCH1 promoter activity by 3.1-fold, whereas a cotransfection with a vector encoding for H19 negated this response (Figure 5B). Similarly, in p53-null Saos2 cells, which do not express H19 (Figure 1G), the transfection of p53 increased NOTCH1 promoter activity by 1.7-fold, whereas transfection of H19 abrogated this response (Figure 5C). In VICs, a knockdown of p53 (Figure 5D) increased the mineralization of cell cultures after treatment with the mineralizing medium for 7 days (Figure 5E). We next hypothesized that H19 may interact with p53 and impede its activity. However, we found that after knockdown of H19 in VICs, the expression of the p53-target genes CDKN1A, mouse double minute 2 homolog (MDM2), and BAX was not modified (Figure 5F–5H). These data thus suggested that H19 does not interact directly with p53. In this regard, the transfection of 293T cells with a vector encoding for H19 containing an S1 aptamer (H19-S1), which can be used in pull-down assay, did not show any significant interaction between H19-S1and p53 (Figure 5I). In addition, the transfection of 293T cells with H19 did not modify the p53 level measured by ELISA (Figure 5J). In the same line, after the immunoprecipitation of p53 in 293T cells, we found that the interaction with MDM2, an E3 ubiquitin ligase and a negative regulator of p53, was not modified by transfection with a vector encoding H19 (Figure 5K). We next hypothesized that H19 may impede the binding of p53 to the NOTCH1 promoter. One p53 consensus site is located in the NOTCH1 promoter region (−850 bp; Figure 5L). In quantitative chromatin immunoprecipitation assay using an antibody for p53, we found that a transfection of H19 in 293T cells reduced the binding of p53 to the promoter region of NOTCH1 by 55% (Figure 5L). In chromatin isolation by RNA purification using H19-S1, we found that H19 was recruited and significantly enriched to the promoter region of NOTCH1 containing the p53 consensus site (Figure 5M). Hence, the recruitment of H19 to the promoter region of NOTCH1 was accompanied by an eviction of p53 from this site and a silencing of NOTCH1.

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Discussion

To the best of our knowledge, this is the first work to report the dysregulation of lncRNA in CAVD. We showed that H19 is overexpressed in CAVD and promotes an osteogenic phenotype in VICs. The level of H19 in human CAVD was associated with different indexes of disease activity such as the progression rate of stenosis. We found that overexpression of H19 was not the result of a loss of imprinting but rather that it was related to a decrease in DNA methylation in the promoter region of this lncRNA. After in-depth functional investigations, we next identified that H19 impeded NOTCH1 expression, which, in turn, resulted in the osteogenic transition of cells (Figure 5N).

Mineralization of the Aortic Valve

Work conducted in the last several years has pointed out that the mineralization of the aortic valve is a key event associated with the development and progression of CAVD.²⁵ Histological analyses of surgically explanted valves showed that mature bone-like structures are present in 10% to 15% of examined valves.^26,27 Studies have also highlighted that several transcripts related to osteogenesis were overexpressed in mineralized, stenotic aortic valves.^6,11 Recently, we reported that single nucleotide polymorphisms and expression quantitative trait loci for RUNX2 were associated with a higher expression of this osteogenic gene in CAVD.⁷ These data thus suggested that RUNX2 is likely an important driver of aortic valve mineralization. Hence, factors that participate in the regulation of RUNX2 may play a significant role into the development/progression of CAVD. In the present work, we found that H19 induced the expression of RUNX2 in cells. Knockdown of H19 in VICs reduced the expression of RUNX2 and BGLAP, a downstream target of RUNX2,²⁸ and decreased the mineralization of cell cultures when grown with a mineralizing medium. In addition, the inhibition of BMP2 with noggin largely reduced H19-induced mineralization of VIC cultures. BMP2 is an important morphogen that drives the mineralization process by enhancing the expression of RUNX2.²⁸ We also found that the knockdown of H19 reduced by 30% the mRNA level of CDH11, which is increased in Notch1^+/− mouse VICs and promotes the mineralization of the aortic valve in mice.^23,29 It has been underlined that CDH11 is a marker of dystrophic and osteogenic mineralization in mouse and porcine VICs.^23,29 Of interest, a recent report by Liang and colleagues³⁰ showed in isolated human mesenchymal stem cells that H19 induced an osteogenic phenotype through a Wnt pathway. Hence, collectively, these data suggested that H19 affected the expression of genes that regulate the mineralization of VICs.

H19 and NOTCH1

In 2005, Garg et al¹³ made the seminal discovery that mutations in NOTCH1 promoted the mineralization of bicuspid aortic valve by altering the expression of RUNX2 and BMP2. Since then, others have also emphasized that NOTCH1 may play a significant role in both bicuspid and tricuspid aortic valve mineralization.^12,14 In this regard, downstream targets of NOTCH1 in the signaling cascade include the hairy family of repressors such as HEY1, which exerts a negative control over the expression of RUNX2 and BMP2.¹³ We found in cell assays that overexpression of H19 decreased the expression of HEY1, suggesting that signaling in the NOTCH1 cascade could be altered. To this effect, in a reporter assay, we documented that H19 decreased significantly the promoter activity of a vector containing RBPJ-responsive elements. RBPJ associates with the NICD, the active intracellular domain of NOTCH1, and promotes the activation of target genes such as HEY1.³¹ Moreover, we documented that knockdown of H19 in VICs increased the mRNA level of NOTCH1, whereas in the reporter assay, transfection of H19 in cells that do not express this lncRNA (293T and Saos2) reduced the activity of the NOTCH1 promoter. The transcription factor p53 is an important regulator of NOTCH1.³² In the Saos2 cell line, which does not express p53, the transfection of a vector encoding p53 increased significantly the activity of NOTCH1 promoter, whereas cotransfection with a vector encoding for H19 abrogated this response. Thus, taken together, these data suggested that H19 controlled the osteogenic fate of cells by altering the transcription of NOTCH1. In this regard, in pull-down assays, we documented that H19-S1was recruited to the promoter region of NOTCH1 containing p53-responsive elements. In the same line, we found in quantitative chromatin immunoprecipitation assay that H19 reduced significantly the recruitment of p53 to the promoter region of NOTCH1. In isolated osteoblasts, p53 is a negative regulator of osteogenic differentiation.³³ Accordingly, in the present work, we found that a knockdown of p53 increased the mineralization of VIC cultures. One study has reported that H19 interacts with p53 and thus could interfere with the transcriptional activity of p53.³⁴ However, after the pull-down assay, we did not find an interaction between H19 and p53. In addition, we determined that the levels of other p53 target genes such as CDKN1A, MDM2, and BAX were not altered by knockdown of H19. It is thus possible that H19 assembles a complex of proteins in a cell- and context-dependent manner. Taken together, these data suggested that H19 is recruited to the promoter region of NOTCH1 where it impedes its expression. It is possible that by steric hindrance H19 prevents the recruitment of p53 to the promoter of NOTCH1. However, we cannot exclude that H19 may also remodel chromatin by bringing key protein complex to the NOTCH1 promoter, which could participate in the silencing of this gene.

Epigenetic Mechanism Related to H19 Overexpression in CAVD

The H19-IGF2 locus is regulated, at least in part, by the ICR, which is located ≈4 kilobase upstream of H19.³⁵H19 is expressed by the maternal allele, whereas IGF2 is paternally expressed. The reciprocal imprinting of H19 and IGF2 is controlled largely by the DNA methylation status of the ICR. According to one model, unmethylated ICR on the maternal allele promotes the recruitment of the insulator protein CCCTC-binding factor, which in turn promotes the interaction of a distal enhancer with the promoter of H19.³⁶ Loss of imprinting with biallelic expression has been shown in different disorders such as in cancers that are associated with higher levels of H19.³⁷ We evaluated in control and mineralized aortic valves the monoallelic or biallelic expression pattern of H19. Using heterozygous patients for polymorphism rs2839704 located in exon 5 of H19, we found that cDNA was consistently, in both control and CAVD tissues, homozygous for this variant, indicating a monoallelic expression pattern for H19. However, we found by using multidimensional genomic profiling that CpG methylation level in the promoter region (−388bp; cg25437674) was inversely related with H19 expression. This was confirmed by pyrosequencing in 58 valves in which we found a lower level of CpG methylation in the promoter region of H19 in mineralized, stenotic aortic valves compared with control nonmineralized valves. These data thus suggested that DNA hypomethylation in the promoter region of H19 may contribute to dysregulation of its expression during CAVD. To this effect, 5-aza-2’-deoxycytidine, which demethylates DNA, increased the expression of H19 by 2.5-fold in isolated cells. However, 5-aza-2’-deoxycytidine globally demethylates the DNA. Hence, further work is necessary to examine factors, acquired or inherited, that alter DNA methylation of the promoter region of H19, particularly in the context of CAVD.

Clinical Impact

There is no medical treatment for CAVD.³⁸ Symptomatic patients with end-stage CAVD need an intervention to replace the aortic valve. These interventions, performed either by open heart surgery or by percutaneous technique, are costly and associated with substantial morbidity/mortality. Hence, the development of a treatment that could be implemented early during the disease process could slow or prevent the progression of CAVD. In this work, we highlighted that a high level of H19 in the aortic valve was associated with a higher level of mineral and a faster progression of CAVD. We also documented in sclerotic valves, which represent the early stage of CAVD, that H19 was already upregulated. Hence, it is likely that early during the process H19 is dysregulated and promotes the osteogenic transition of VICs. Further work to understand molecular processes that fine-tune the expression of genes related to the mineralization of the aortic valve could lead to new therapies by regulating H19 lncRNA. To this effect, antisense oligonucleotides³⁹ or drugs that modify the epigenome could be used to reduce the expression of H19.⁴⁰

Limitations

This work was performed with in vitro assays to document the role of H19 on the osteogenic phenotype of cells. Whether H19 promotes the development of CAVD in preclinical animal models remains to be established. However, transgenic overexpression of H19 in mice is lethal during late gestation.⁴¹ Nonetheless, the present findings in human tissues and cell assays generate novel hypotheses about the role of H19 in CAVD.

Conclusions

We found that DNA hypomethylation in the promoter region of H19 is associated with a higher level of this lncRNA during CAVD. Moreover functional assays indicate that H19 is a novel regulator of the osteogenic fate of cells by regulating the expression of NOTCH1.

Footnotes