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H19 Induces Abdominal Aortic Aneurysm Development and Progression

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.117.032184Circulation. 2018;138:1551–1568

Abstract

Background:

Long noncoding RNAs have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize long noncoding RNAs as potential mediators in abdominal aortic aneurysm development.

Methods:

We profiled RNA transcript expression in 2 murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in apolipoprotein E–deficient (ApoE−/−) mice (n=8) and porcine pancreatic elastase instillation in C57BL/6 wild-type mice (n=12). The long noncoding RNA H19 was identified as 1 of the most highly upregulated transcripts in both mouse aneurysm models compared with sham-operated controls. This was confirmed by quantitative reverse transcription–polymerase chain reaction and in situ hybridization.

Results:

Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo, significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human abdominal aortic aneurysm tissue samples, and in a novel preclinical LDLR−/− (low-density lipoprotein receptor) Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, whereas overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1α as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and hypoxia-inducible factor 1α and sequential p53 stabilization. Additionally, H19 induced transcription of hypoxia-inducible factor 1α via recruiting the transcription factor specificity protein 1 to the promoter region.

Conclusions:

The long noncoding RNA H19 is a novel regulator of SMC survival in abdominal aortic aneurysm development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.

Clinical Perspective

What Is New?

  • Development, progression, and rupture of abdominal aortic aneurysms remain a major problem in patient health care.

  • Apart from surgical and interventional treatment strategies, no pharmacological therapeutic option exists that could halt aneurysmal expansion.

  • Here, we identify the long noncoding RNA H19 with functional relevance in experimental aortic aneurysm progression in 2 murine models, a novel preclinical mini-pig model utilizing genetically mutated mini-pigs, as well as end-stage human disease.

  • H19 mediates expression levels of the transcription factor hypoxia-induced factor 1α, which in the chronic hypoxic environment of an aneurysm triggers apoptosis in aortic smooth muscle cells.

What Are the Clinical Implications?

  • Specific inhibition of noncoding RNAs (such as microRNAs or long noncoding RNAs) using antisense oligonucleotides is providing us with new options for disease treatment.

  • Several clinical studies are currently ongoing that investigate the potential of these RNA-based therapies.

  • Our work introduces inhibition of H19 with GapmeRs as a novel molecular therapy to limit smooth muscle cell death in progressing aortic aneurysms.

  • Increasing smooth muscle cell survival in aneurysms through this treatment strategy limits aortic dilatation, and essentially the need for surgical intervention, as well as the risk of acute ruptures that bear high mortality rates.

Introduction

Abdominal aortic aneurysms (AAAs) are localized enlargements of the abdominal aorta with diameters >3 cm, or >1.5 times their normal size.1 AAA are most commonly found in the infrarenal abdominal aorta, with an overall prevalence of 6% in men and 1.6% in women.2 The asymptomatic characteristic of aneurysms makes their assessment challenging. Unless AAAs rapidly increase in size, acutely rupture, or thromboembolize into the distal arterial system, they typically remain clinically silent. Fatal outcome attributable to rupture occurs when intraluminal pressure and wall stress exceeds the strength of the aortic wall, with mortality rates as high as 80%.3 It is estimated that ruptured AAAs account for nearly 15 000 deaths in the United States annually.4 Unfortunately, no effective pharmacological approach has been identified to date that can limit their progression or the risk of rupture in humans.5 Clinically, the only available treatment option remains surgical intervention, which includes open or endovascular aortic repair of the dilated aorta.5 Obtaining a better understanding of the cellular mechanisms and regulatory networks driving AAA development and progression is essential to identifying novel therapeutic targets.

Emerging evidence has shown that noncoding RNAs (ncRNAs), including microRNAs (miRNAs), that consist of 22 nucleotides (nt), and long noncoding RNA (lncRNA) >200nt in length, function as powerful mediators in all aspects of molecular regulation under physiological and pathological conditions.6,7 Our previous studies have identified several miRNAs (miR-21,8 −29b,9 −2410) as key factors and potential therapeutic targets in experimental murine models of AAA development and progression, as well as human disease. The contribution of lncRNAs to AAA expansion and rupture has yet to be experimentally addressed. The aim of the current study was to evaluate whether—and how—lncRNAs are involved in this process.

We used RNA profiling of experimental murine models of AAA disease, a novel preclinical Ldlr−/− (low-density lipoprotein receptor) Yucatan mini-pig model, as well as human tissue samples from patients with stable and ruptured AAA disease (versus organ donor controls) and discovered increased levels of the lncRNA H19 during AAA development and expansion. H19 is capable of mediating expression levels, cellular localization, and functionality of hypoxia-induced factor 1α (HIF1α), which exerts proapoptotic and anti-proliferative effects in aortic smooth muscle cells (SMCs) leading to subsequent aortic dilation.

Methods

The data, analytic methods, and study materials will be available to other researchers for purposes of reproducing the results or replicating the procedure.11

Murine AAA Model

All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University (http://labanimals.stanford.edu/) or the Karolinska Institutet, and followed the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animals were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were performed with 10-week-old male C57BL/6J (porcine pancreatic elastase [PPE]10) and 10-week-old male apolipoprotein E–deficient (ApoE−/−) C57BL/6J mice (Angiotensin [Ang] II). A detailed description is provided in the online-only Data Supplement.

PPE Infusion in Ldlr−/− Yucatan Mini-Pigs

One-year-old Yucatan mini-pigs (Ldlr−/− or Ldlr+/+)12 were provided by Exemplar Genetics (Sioux Center, IA). The experiments in mini-pigs were approved by the Ethics Board of Northern Stockholm. In a total of 12 genetically mutated mini-pigs, AAAs were induced using the PPE instillation method similar to studies described in mice in our current manuscript, and as previously described by others in landrace pigs.13 A detailed description is provided in the online-only Data Supplement.

LNA–Anti-H19 GapmeR Injection

Either an LNA–anti-H19 GapmeR or scr-GapmeR (LNA negative control GapmeR from Exiqon) was injected intraperitoneally (in 0.2 mL of PBS over 3–6 seconds). The concentration of anti-GapmeR or scrambled GapmeR was 20 mg/kg. Injections were carried out at 0, 7, and 14 days after AAA induction in both murine AAA models. The custom-made LNA–anti-H19 GapmeR sequence was 5’-GACGGAGATGGACGAC-3’. The LNA scrambled GapmeR control sequence was 5’-GTGTAACACGTCTATACGCCCA-3’.

Aortic Diameter Measurements by Ultrasound Imaging

At baseline, and 7, 14 and 28 days after aneurysm induction, B-mode ultrasound imaging was performed to assess the abdominal aortic diameter. For the detailed process, please refer to the Expanded Methods in the online-only Data Supplement.

RNA Quantification and Gene Expression

Total RNA was isolated with a TRIzol-based (Invitrogen) RNA isolation protocol. Gene expression was quantitatively analyzed using Taq-Man quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assays. For the detailed process, please refer to the online-only Data Supplement.

RNA Sequencing Data Analysis

Raw reads from 4 saline and 4 AngII-treated ApoE−/− mice harvested at 7 days were aligned to the GRCm38 mouse reference genome with STAR14 using default parameters. The complete dataset has been uploaded to the Gene Expression Omnibus. Detailed procedures are explained in the online-only Data Supplement.

RNA Profiling (Microarray) and Data Analysis

RNA profiling was performed using the Mouse Transcriptome Assay (Affymetrix). The Affymetrix transcriptome analysis console v3.0 was used for differential expression analysis, according to the manufacturer’s instructions. The final analysis included 7-day PPE-treated aortae (n=6) versus controls (sham-operated, saline-treated, n=6). A false detection rate adjusted P value <0.05 was considered significant. The complete dataset has been uploaded into the Gene Expression Omnibus.

Histological, Immunohistochemical, and Immunofluorescent Analysis

Histological staining after harvesting was performed in the same region of the abdominal aorta that was imaged to obtain morphometric data to correlate with ultrasound measurements and gene expression results from qRT-PCR using a standardized protocol as previously described.15 Detailed procedures are explained in the online-only Data Supplement.

Combined In Situ Hybridization/Proximity Ligation Assay

In situ hybridization (ISH

) was performed as described above, utilizing the miRCURY LNA microRNA ISH Optimization Kit (Exiqon; see the online-only Data Supplement) and 5’-Biotin labeled probes for H19 or the HIF1α promoter region. After the ISH procedure, the Duolink assay (Sigma-Aldrich) was initiated with overnight incubation of a mouse antibody against biotin and rabbit antibodies targeting HIF1α or specificity protein 1 (Sp1; Abcam). The procedure was continued the next day according to the manufacturer’s instructions and slightly modified for RNA-protein interactions.16 The amplified interaction signal was captured and analyzed using a Leica Confocal Microscope.

In Vitro Cell Culture and Transfection

Human aortic SMC (hAoSMCs) were propagated in growth media (SmGM-2; Lonza) with 5% fetal bovine serum per standard protocols (Lonza; passage no. 4–5). Lipofectamine RNAiMAX (Invitrogen) reagent was used for transfection of small oligonucleotides, and Lipofectamine 3000 (Invitrogen) was used for vector transfection. For detailed process, please refer to the online-only Data Supplement.

Kinetic Assessment of Proliferation and Apoptosis in hAoSMCs

The IncuCyte Zoom System (Essen Biosciences) was used for real-time and dynamic assessment of the hAoSMCs status over time17. For detailed process, please refer to the online-only Data Supplement.

Cell Fractionation and Immunoblotting

Cytoplasmic and nuclear fractions were isolated using the PARIS kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Western blotting was carried out as previously described18 (online-only Data Supplement). Tubulin was used as cytoplasmic loading control, and Lamin B1 as the nuclear loading control.

Luciferase Reporter Assay

Transcriptional activity of HIF1α was confirmed by transfecting hAoSMCs with pGL-HIF1A prom luciferase vector (Addgene), with or without recombinant Sp1 protein (Abcam)/AngII treatment and pcDNA-H19 wt/deletion. Luciferase activity, 24 hours after treatment, was determined using the Lightswitch Assay (Switchgear) as previously described.19

Chromatin Immunoprecipitation

Occupancy of Sp1 in the HIF1α promoter region was evaluated via a chromatin immunoprecipitation assay20. For the detailed process, please refer to the online-only Data Supplement.

Human Tissue Sample Acquisition and Preparation

Human samples from the Munich Vascular Biobank from patients who underwent surgical repair of their AAA (n=20), as well as abdominal aortic samples from organ donor controls (n=10), were harvested during surgery (or explantation), snap-frozen, and stored at −80°C. Approval for studies on human tissue samples was obtained under informed consent, complying with all guidelines and policies at the Klinikum rechts der Isar, Technical University Munich, and in accordance with the Declaration of Helsinki. Patient samples were matched for age (72.3 on average), sex (only males), cardiovascular risk factors (smoking: all smokers, diabetes mellitus: all nondiabetics, without known medical therapy), dyslipidemia (total cholesterol <200 mg/dL; triglycerides < 185 mg/dL), and medication (no antiplatelet or anticoagulant therapy, all patients on statin therapy).

Statistics

Data are presented as mean±SEM, unless stated differently. Groups were compared using Student t test. Normality was tested to ensure that parametric testing was appropriate. When comparing multiple groups, data were analyzed using ANOVA with Bonferroni post-test. Sequential measurements (abdominal aortic diameters at consecutive time points) were analyzed by two-way repeated-measures ANOVA. Categorical data were analyzed by using the χ2 test. A value of P<0.05 was considered statistically significant.

Results

H19 Is Significantly Upregulated in AAA Models, and Knockdown of H19 Limits Murine AAA Growth

We performed RNA sequencing in the AngII/ApoE−/− mouse model to identify dysregulation during aneurysm development. Abdominal aortic tissue from either AngII-treated mice (n=4) or sham controls (n=4) were harvested 7 days after pump implantation. The lncRNA H19 was identified as one of the most substantially upregulated transcripts in AAA tissue (Figure 1b). When cross-checking upregulated transcripts in a second experimental AAA mouse model using PPE infusion, H19 and 2 other lncRNAs (A530020G20Rik, F630028O10Rik) were identified as model-independent lncRNAs with potential implications for experimental AAA progression on analysis of a mouse transcriptome array. Of further importance, all 3 transcripts have human homologues, allowing us to evaluate their relevance and potential changes in AAA samples of human origin (Figure 1a and 1b). Upregulation of all 3 lncRNAs was confirmed by RT-qPCR in AngII-induced AAA samples versus controls (Figure 1c). H19 was chosen for further investigations, as it was the most significantly upregulated IncRNA in mouse and human samples compared to the other lncRNAs and their homologues.

Figure 1.

Figure 1. H19 is significantly upregulated in abdominal aortic aneurysm (AAA) models and knockdown of H19 limits AAA growth. A, Aortic tissue was collected from 2 AAA models; firstly, 10-week-old ApoE−/− male mice were either implanted with osmotic pumps containing Angiotensin II (AngII; 1μg/kg/min) or phosphate buffered saline (PBS). Secondly, porcine pancreatic elastase (PPE)-induced AAA was performed in 10-week-old male C57BL/6J mice. AAAs and controls were subjected to RNA sequencing (AngII) or microarray (PPE) analysis. B, Volcano plot of deregulated transcripts in the AngII-AAA model (7 days postinduction). C, Reverse transcription–polymerase chain reaction (qRT-PCR) validation of upregulated and relevant lncRNAs (cross-checked with PPE microarray dataset) in the AngII-AAA. D, Relative diameter of aortas at baseline, and after 7, 14, and 28 days post aneurysm induction, B-mode ultrasound imaging was performed to assess the aortic aneurysm diameter (AAD). Data are presented as growth fold change and analyzed using 2-way repeated measures ANOVA. In vivo knockdown of H19 utilizing site-specific antisense oligonucleotides (LNA-GapmeRs, H19 KD) limits aneurysm progression. AngII-AAA mice were intraperitoneally injected with H19 GapmeR (H19 KD, n=6; 20 mg/kg bodyweight) or scrambled oligo controls (SCR, n=7; 20 mg/kg bodyweight). E, Representative images of aneurysmal (treated with either SCR or H19 KD) and undiseased, sham-infused aortas (NC). F, In situ hybridization of H19 (purple) and immunochemical staining of smooth muscle cell a-actin (SMA, brown). H19 expression correlated with SMC content. *P<0.05 vs 0 d, #P<0.05 vs SCR, **P<0.01 vs saline sham control. Bar, 100 μm.

The incidence, growth rate, and size of aneurysmal expansion was measured by ultrasound at baseline (day 0) and after 7, 14, and 28 days post-AAA induction (Figure Ia in the online-only Data Supplement). Of importance, systemic in vivo knockdown of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs; Figure Ib, Ic, and Id in the online-only Data Supplement), significantly limited aneurysm growth in both murine models (Figure 1d and 1e; Figure IIa in the online-only Data Supplement). Mismatched, scrambled oligonucleotides were used as relevant controls. Fluorescent imaging of fresh aortic sections indicated sufficient transvascular distribution of the prelabeled GapmeR (Figure Ic in the online-only Data Supplement). Knockdown of H19 was confirmed with qRT-PCR in various organs in addition to the abdominal aorta (Figure Id in the online-only Data Supplement).

In situ hybridization indicated that H19 is expressed in both medial SMCs and adventitial fibroblasts of the undiseased, nondilated aorta (Figure 1f: NC). On AngII infusion, H19 expression was substantially elevated in medial SMCs in dilated aortic tissue (Figure 1f). This increase was abolished by inhibiting H19 with GapmeRs. These data suggest that H19 participates in AAA development and progression possibly through influencing SMC dynamics.

H19 Regulates Smooth Muscle Cell Function

Consistent with our in vivo data (Figure 1b and 1c), AngII was able to robustly induce H19 in cultured aortic SMCs (Figure IIIa in the online-only Data Supplement). AngII treatment further induced apoptosis, while decreasing proliferation rates of SMCs (Figure IIIb through IIId in the online-only Data Supplement). These effects were both attenuated by H19 knockdown (Figure IIIb through IIId in the online-only Data Supplement), indicating the regulatory role of H19 in SMC apoptosis under AAA-relevant (AngII) conditions. Of note, this proapoptotic effect could only be observed at a later timepoint (48 h), and not during the early, acute response phase (8 h; Figure III in the online-only Data Supplement).

To further evaluate the cellular effects of H19 modulation in SMCs, we used a kinetic live cell imaging system. First, we either inhibited H19 with LNA-GapmeRs (Figure 2a) or induced H19 with pcDNA-H19–dependent overexpression (Figure 2b) in cultured hAoSMCs. Two different custom anti-H19 GapmeRs were tested for knockdown efficiency in hAoSMCs, and the more potent one was chosen for further studies (and was used for the in vivo experiments; Figure Ib in the online-only Data Supplement). Cells were kinetically monitored for rates of proliferation and apoptosis. Overexpression of H19 (Figure 2b) resulted in enhanced apoptosis (Casp3+, green dots) and decreased proliferation (confluence %, orange mask) of SMCs in a time-dependent manner (Figure 2c through 2e). Knockdown of H19 (Figure 2a) inhibited SMC apoptosis, whereas no significant effect on the proliferation rate could be observed (Figure 2c through 2e; Movies 1 through 4 in the online-only Data Supplement).

Figure 2.

Figure 2. H19 promotes apoptotic cell death in vascular smooth muscle cells. Reverse transcription–polymerase chain reaction (qRT-PCR) validation of H19 modulation using (A) LNA-GapmeRs (H19 KD) or (B) overexpression with the pcDNA-H19 vector (H19 OE). SMC apoptosis (Casp3+; C) and proliferation (confluence in %; D) were assessed via kinetic live cell imaging over time. E, Representative images of proliferation (orange mask) and apoptosis (green dots) from the kinetic live-cell imaging system. *P<0.05 vs SCR or NC, **P<0.01 vs SCR or NC. Bar, 100 μm.

H19 Effects on Smooth Muscle Cells Are Independent of miR-675

Accumulating studies have suggested that H19 may function as a reservoir of miR-675, which is embedded in the first exon of the H19 transcript.21,22 Thus, we evaluated the role of miR-675 in H19-mediated effects on SMCs. Cultured hAoSMCs were transfected with either H19 wild-type vector (H19 wt) or H19 mutant with deletion of miR-675 (H19 mut, Figure 3a), and monitored over time. Both H19 mut and H19 wt induced apoptosis and reduced proliferation of SMCs (Figure 3b and 3c). Apart from this, no significant difference in miR-675 expression was observed in AngII-induced AAAs by qRT-PCR (Figure 3d). Unlike H19, miR-675 was furthermore not significantly deregulated in tissue samples from human patients with AAA, or in either of the 2 experimental murine models of aneurysm development. Thus, the effects of H19 on SMCs appear independent of miR-675 during AAA development.

Figure 3.

Figure 3. H19 induces smooth muscle cell apoptosis via hypoxia-induced factor 1α (HIF1α). A, Schematic graph of the deletion of miR-675 in the first exon of H19 (H19 mut). Smooth muscle cell (SMC) apoptosis (Casp3+; B) and proliferation (confluence in %; C) after transfection of H19 wt or mut were assessed via kinetic live cell imaging over time. D, Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) measurement of H19 and miR-675 in angiotensin (Ang) II-abdominal aortic aneurysms (AAAs). E, Predicted panel of transcription factors potentially interacting with H19, exploiting a binding free energy below −30 Kcal/mol. F, qRT-PCR measurement of the panel of transcription factors on H19 knockdown (H19 KD) or overexpression (H19 OE). G, Immunofluorescent staining of HIF1α (green) and Caspase 3 (red) on AngII treatment, modulation of H19 or HIF1α, or combined treatment. H, qRT-PCR measurement of genes on AngII treatment, overexpression of HIF1α—with or without knockdown of p53. I, Immunofluorescent staining of Caspase 3 (red) on AngII treatment, overexpression of HIF1α—with or without knockdown of p53. J, Validation of HIF1α–Mdm2 interaction using immunoprecipitation Western blot. K, Protection of AngII treatment or overexpression of HIF1α on Mdm2-mediated reduction of p53. *P<0.05 vs SCR or NC, **P<0.01 vs SCR or Saline; #P<0.05 vs AngII or HIF1α OE; Bar, 50 μm.

H19 Induces Smooth Muscle Cell Apoptosis via HIF1α

Transcription factors have been well recognized as master regulators controlling gene expression, chromatin stability and cell homeostasis.23–25 To explore how H19 regulates SMC apoptosis, we proposed that H19 might affect molecular processes via alteration of transcription factors. Based on an in silico analysis of regulatory RNA elements,26 we identified several transcription factors that were predicted to interact with H19, but had not been experimentally validated. The analysis concluded that cAMP responsive element binding protein 1 (CREB1), transcription factor CP2 (TFCP2), GTF2I repeat domain containing 1 (GTF2IRD1), SMAD family member 4 (SMAD4), HIF1α, zinc finger and BTB domain containing 14 (ZBTB14), Krüppel-like factor 11 (KLF11), and transcription factor AP-2 alpha (TFAP2α) are possibly involved in the molecular function of H19. All of these transcription factors had a predicted binding free energy of <−30 Kcal/mol (Figure 3e).

First, to determine whether these transcription factors are regulated on the RNA level as part of a feed-forward loop, a custom-designed RNA array was used to investigate changes in transcription factor expression in SMCs on overexpression and knockdown of H19. Results revealed that HIF1α was the only differentially regulated factor (Figure 3f). In addition, HIF1α protein levels decreased after H19 knockdown and concomitantly increased after vector-based H19 induction (Figure IVa in the online-only Data Supplement).

To confirm that HIF1α mediates the effects of H19, we used siRNA-induced inhibition of HIF1α. Both mRNA (Figure 4b in the online-only Data Supplement) and protein (Figure 4c in the online-only Data Supplement) levels of HIF1α were significantly blocked. Interestingly, H19 overexpression as well as AngII-treatment dramatically increased the expression of HIF1α and consequently apoptosis in SMCs (Figure 3g, upper panel). This effect could be abolished by inhibition of H19 or HIF1α silencing (Figure 3g, bottom panel and right panel). H19 levels in SMCs remained unchanged on HiF1α inhibition (Figure 4d in the online-only Data Supplement). Importantly, the proapoptotic protein Bcl-2 associated X (BAX) displayed the same pattern as HIF1α, whereas the antiapoptotic mediator B-cell CCL/lymphoma 2 (BCL2) showed the opposite trend (Figure 4e in the online-only Data Supplement), suggesting that AngII/H19-induced SMC apoptosis is dependent on changes in HIF1α expression.

To further dissect the mechanism through which HIF1α triggers SMC apoptosis, we either performed HIF1α overexpression or induced its expression via the AAA-related factor AngII. In both cases, BAX expression and the SMC apoptotic ratio substantially increased. This could be attenuated by p53 knockdown (Figure 3h and 3i). It was recently reported that BAX is regulated by p5327 and has multiple p53 binding sites in its promoter region (Figure 4f in the online-only Data Supplement). Previous studies have shown that p53 is controlled by the pro- to-oncogene transformed mouse 3T3 cell double minute 2 (Mdm2)28, a ubiquitin E3 ligase, via a feedback mechanism in lung cancer cells and mouse embryonic fibroblasts.17 p53 is an established factor that triggers aneurysm expansion.29,30 We were able to confirm that HIF1α directly interacts with Mdm2, preventing Mdm2-mediated reduction of p53 (Figure 3j and 3k). This suggests that HIF1α induces SMC apoptosis via regulating Mdm2-p53 activity during aortic dilatation.

H19 Induces Smooth Muscle Cell Apoptosis via HIF1α in Preclinical Models of AAA and Is Relevant to Human Disease

To confirm that HIF1α contributes to H19-induced SMC apoptosis and is relevant in AAA, we evaluated the expression of HIF1α and apoptosis-related genes in both mouse and human AAA samples with qRT-PCR. As expected, H19, HIF1α, and BAX were all significantly increased, whereas the antiapoptotic protein BCL2 was markedly decreased in the murine AngII and PPE models, as well as in human aortic aneurysms (Figures 4a and 5a; Figure IIb in the online-only Data Supplement). Utilizing immunohistochemical analysis, we detected HIF1α upregulation in medial SMCs, correlating with cell apoptosis in both experimental AAA models (Figure 4c: SCR; Figure IIc through IIe in the online-only Data Supplement: SCR). Depletion of H19 sufficiently suppressed the expression of HIF1α and significantly limited SMC apoptosis (Figure 4b and 4c: H19 KD; Figure IId and IIe in the online-only Data Supplement: H19 KD). Again, colocalization of upregulated H19 and HIF1α, as well as a correlation with cellular apoptosis, was observed in SMC layers of human AAA specimens (Figure 5b, Figure V in the online-only Data Supplement). H19 and HIF1α were consistently induced in both murine AAA models, whereas specific changes for known HIF1α downstream targets were inconsistent in the different AAA models (Figure Vc in the online-only Data Supplement), indicating that these changes are likely to be model-related rather than HIF1α-dependent.

Figure 4.

Figure 4. H19 inhibition reduces smooth muscle cell apoptosis via hypoxia-induced factor 1α (HIF1α) in angiotensin (Ang) II–induced abdominal aortic aneurysms (AAAs). A, Reverse transcription–polymerase chain reaction (qRT-PCR) measurement of H19, HIF1α, BAX, and BCL2 in AngII-induced AAA. B, Quantification of HIF1α protein expression via Western Blot (n=3). C, Immunochemical staining of smooth muscle cell a-actin (SMA, red), HIF1α (brown), and Caspase 3 (red). *P<0.05, **P<0.01 vs saline. Bar, 100 μm.

Figure 5.

Figure 5. H19 induces smooth muscle cell apoptosis via hypoxia-induced factor 1α (HIF1α) in preclinical models of abdominal aortic aneurysms (AAAs) and is of relevance to human disease. A, Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) detection of H19, HIF1α, BAX and BCL2 expression levels in human AAAs. B, Immunochemical staining of smooth muscle cell a-actin (SMA, red) and Caspase 3 (red); immunofluorescence staining of HIF1α (green), and fluorescent in situ hybridization of H19 (red). C, Growth curve of aortic aneurysm diameter (AAD) in Ldlr−/− Yucatan mini-pigs on porcine pancreatic elastase (PPE) instillation or sham operation (heat-inactivated PPE) analyzed by 2-way repeated measures ANOVA. D, qRT-PCR measurement of H19 and HIF1α in Ldlr−/− Yucatan mini-pigs on PPE instillation or sham operation. Morphology of pig PPE-AAA and sham with hematoxylin & eosin (H&E; E) and Elastica van Giesson (EvG; F) staining. Highlighted areas indicate aneurysm-related stretching, hyperplasia, and breakdown of elastic layers. *P<0.05, **P<0.01 vs NC; #P<0.05 vs Ldlr−/− sham. Bar, 50 μm.

Finally, AAA was induced in wild-type (WT) or Ldlr−/− Yucatan mini-pigs through PPE perfusion, similar to the murine model. The incidence, growth rate, and absolute size of aneurysmal expansion were measured by ultrasound at baseline, and at 7 and 28 days after AAA induction (Figure 5c). Compared with the sham-operated group, the expression of H19 was significantly upregulated along with enhanced expression of HIF1α in PPE-induced AAAs (Figure 5d and 5e).

H19 Detains HIF1α in the Cytoplasm via Direct Interaction

Cultured hAoSMCs subjected to either H19 modulation or treatment with AngII underwent ISH, immunofluorescent staining, and proximity ligation assay. The analysis indicated that both AngII treatment and H19 overexpression resulted in increased expression of HIF1α and colocalization with H19, whereas knockdown of H19 reduced HIF1α (Figure 6a).

Figure 6.

Figure 6. H19 detains hypoxia-induced factor 1α (HIF1α) in the cytoplasm via direct interaction. A, Immunofluorescent staining of HIF1α (green), and fluorescent in situ hybridization of H19 (red) after AngII treatment, H19 overexpression (H19 OE), or H19 knockdown (H19 KD). B, RegRNA in silico predicted secondary structure of H19 interacting with the HIF1α transcription factor (protein), using a calculated binding free energy of −33.9 Kcal/mol. C, H19 was detected using a biotin labeled in situ probe, followed by a proximity ligation assay targeting HIF1α. The positive red signal indicates the interaction of H19 with HIF1α. D, Cell fractions were isolated and measured with a Western blot detecting HIF1α. LaminB (cytoplasm) and tubulin (nucleus) were used as loading controls. Histogram depicts relative HIF1α levels as fold change vs the respective cytoplasmatic or nuclear loading control. **P<0.01 vs NC, #P<0.05 vs H19 CoCl2. Bar, 50 μm.

Colocalization only indicates closeness of different components, and fluorescence microscopy is insufficient to identify the physical apposition of 2 molecules.31 Bioinformatic analysis26 revealed the secondary structure of a HIF1α binding site on H19, with a low binding free energy of −33.9 Kcal/mol (Figure 6b). To assess the physical interaction between H19 and HIF1α, we used ISH and proximity ligation assay, in which amplified signals can be detected once 2 molecules reach close proximity (<40 nm), considered proof of their interaction,32 Indeed, AngII treatment as well as H19 overexpression substantially enhanced the interaction between H19 and HIF1α, predominantly in the cytoplasm (Figure 6c).

HIF1α is required for embryonic cardiovascular development, and thus is considered essential for cell survival during hypoxia. During this process, induced HIF1α translocates into the nucleus and activates transcription of many genes involved in the attenuation of the hypoxic damage.33 Cobalt chloride (CoCl2), a well-established chemical inducer of HIF1α,34 successfully increased accumulation of HIF1α and triggered its translocation into the nucleus (Figure 6d). Interestingly, H19 attenuated the translocation and detained HIF1α within the cytoplasm, suggesting that this direct interaction of H19 binding cytoplasmic HIF1α can stimulate SMC apoptosis and enhance AAA progression.

H19 Increases Nuclear HIF1α Transcription via Recruitment of Sp1

To further dissect the molecular mechanism of HIF1α regulation by H19, we assessed the transcriptional activity of HIF1α. It has previously been shown that HIF1α transcription is dependent on the transcription factor Sp1,35,36 with multiple binding sites located in the HIF1α promoter (Figure 7a). Thus, we investigated whether H19—apart from its regulatory role in the cytoplasm—can also directly influence HIF1α transcription by interacting with Sp1-mediated effects in the promoter region of the HIF1α gene. We designed a biotin-labeled ISH probe, targeting the HIF1α promoter region, and used the proximity ligation assay-based interaction detection method as described above. Under control conditions (using a scrambled probe), only weak Sp1 binding to the HIF1α promoter was observed (Figure 7b). The interaction became drastically enhanced on AngII treatment (Figure 7b), as well as on overexpression of H19 (Figure 7b). A diminished effect was seen when performing a proof-of-concept study with knockdown of H19 (Figure 7b). The average signal intensity is summarized in Figure 7c. A similar interaction pattern between Sp1 and H19 was confirmed in the nucleus (Figure VIa through VIc in the online-only Data Supplement). In addition, no significant changes were seen in the expression level of Sp1 in cells on modulation of H19 or AngII treatment, as well as in our murine AAA models (Figure VId in the online-only Data Supplement).

Figure 7.

Figure 7. H19 increases hypoxia-induced factor 1α (HIF1α) transcription via recruitment of specific protein 1 (Sp1). A, Multiple binding sites of the Sp1 transcription factor are predicted in the HIF1α promoter regio (retrieved from the eukaryotic promoter database). B, The HIF1α promoter region is detected by a biotin labeled in situ probe, followed by a proximity ligation assay targeting Sp1. The positive red signal represents the interaction of Sp1 with the HIF1α promoter. C, Quantification of the images presented in B. D, Luciferase activity of the HIF1α promoter representing the transcriptional activity of HIF1α. With or without Sp1 inhibitor (mithramycine A, 1 μmol/L) treatment, cultured hAoSMCs transfected with a luciferase vector containing the HIF1α promoter wt, and stimulated with H19 overexpression (H19), recombinant Sp1 protein (Sp1), both H19 overexpression and Sp1 (H19+Sp1), Angiotensin II (AngII), and compared with normal control (NC). E, Luciferase activity of the HIF1α promoter representing the transcriptional activity of HIF1α. Cultured hAoSMCs cells transfected with a luciferase vector, containing the HIF1α promoter wt or different mutants (M1-M5), and stimulated with H19 overexpression (H19) or Angiotensin II (AngII), and compared with normal control (NC). F, Schematic graph of the deletion of the H19 binding site with the HIF1α promoter (predicted by LongTarget). G, Luciferase activity of the HIF1α promoter after treatment with a H19 deletion mutant (H19 del), recombinant Sp1 protein (Sp1), or in combination (H19 del+Sp1). Stimulated (AngII) and H19-modulated smooth muscle cells were subjected to chromatin immune precipitation (ChIP) with Sp1 antibodies, followed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) using specific probes targeting the HIF1α promotor region, or −10kb upstream (H) or H19 (I), respectively. *P<0.05, **P<0.01 vs NC or SCR, #P<0.05 vs Sp1 or H19; &P<0.05 vs HIF1α prom wt; Bar, 50 μm.

To assess the transcriptional activity, we then used a luciferase reporter assay containing the HIF1α wt or Sp1-binding site mutants in the promoter region. Cultured hAoSMCs were transfected with a pGL-HIF1α promoter luciferase vector, with or without the recombinant Sp1 protein/AngII treatment and pcDNA-H19 wt/deletion. Sp1 treatment clearly led to an induction of luciferase activity, representing the transcriptional activity of HIF1α, which served as a positive control (Figure 7d). Overexpression of H19 or AngII treatment displayed similar effects (Figure 7d). The combined treatment of Sp1 and H19 overexpression substantially increased transcriptional activity, suggesting that H19 recruits and facilitates Sp1 binding to the HIF1α promoter region (Figure 7d). These effects were abolished in the presence of the Sp1 inhibitor mithramycine A, further supporting the transcriptional role of Sp1 in this scenario (Figure 7d). In addition, luciferase assays of HIF1α promoter mutants (M1-M5) indicated 2 Sp1 binding sites (−66 and −79) in the HIF1α promoter region as being essential for these effects (Figure 7e). Moreover, analyzing the sequence of H19 and HIF1α promoter (+500 to −2000 bp) using the LongTarget tool,37 a H19-HIF1α binding pattern was predicted as shown in Figure 7f. Deletion of this region in H19 (H19 del) retained proapoptotic capacity (Figure VIe in the online-only Data Supplement) but failed to enhance HIF1α transcriptional activity as H19 wt did (Figure 7g). Taken together these data suggest that H19 enhances HIF1α transcription via recruitment of Sp1 to its promoter region.

Finally, a chromatin immune precipitation assay was able to confirm the occupancy of Sp1 and H19 in the HIF1α promoter. SMCs were subjected to chromatin immune precipitation with Sp1 antibodies, followed by qRT-PCR measurements with specific probes targeting the HIF1α promoter or −10kb upstream (control), or H19 respectively. The results indicated that the occupancy of Sp1 in the HIF1α promoter region was significantly increased compared to that of the −10kb upstream/control (Figure 7h) in response to treatment with AngII or H19 overexpression, but not on H19 knockdown. Similar trends were observed for H19 levels (Figure 7i). In addition, H19 was significantly increased with Sp1 RNA-chromatin immune precipitation on stimulation with AngII or H19 overexpression, further validating the direct interaction between Sp1 and H19 (Figure VIf in the online-only Data Supplement).

Discussion

LncRNAs are defined as transcripts longer than 200nt without evident protein coding function. Recent studies have indicated their involvement in numerous biological processes and disease development, such as promoting or inhibiting transcription and translation, scaffolding regulatory proteins, shaping the nuclear architecture, imprinting genomic loci, or acting as enhancers.7,38,39 However, the expression of many lncRNAs is restricted to isolated developmental contexts, specific species, or aberrantly regulated diseases.40,41 Thus, for experimental investigations and translational purposes, we decided to focus on lncRNAs that are functionally conserved in mice and humans with potential implications for AAA disease. By performing RNA profiling in 2 different murine AAA models, we identified 3 differentially expressed lncRNAs with human homologues. H19, with a previously unknown contributory role in AAA disease, was the most consistently upregulated transcript.

H19 was initially reported as a maternally imprinted ncRNA that remains highly expressed throughout embryonic and fetal development. Apart from the placenta, it was shown that its expression is shut down in most tissues shortly after birth.42 This onco-fetal behavior pattern and its uniparental monoallelic expression have been identified as principal characteristics of imprinted genes. Most of these genes are known to become altered in various malignancies.43 Accumulating data have revealed that these oncogenic properties of H19 are tightly correlated with nullification, dysfunction, or significant downregulation of the master tumor suppressor gene p53.44

In the cardiovascular system, H19 appears to have universal effects. The downregulation of H19 was shown to promote cell proliferation and inhibit cell apoptosis during late-stage cardiac differentiation, by regulating the negative role of miR-19b on Sox6 expression.45 H19 was further found to mediate the inhibition of melatonin by inducing the premature senescence of c-kit+ cardiac progenitor cells via miR-675 stimulation.46 In addition, H19 inhibition was found to decrease human umbilical vein endothelial cell growth and capillary formation,47 whereas the H19-miR-675 axis targets calcium/calmodulin dependent protein kinase II Delta (CaMKIIδ), serving as a negative regulator of cardiac hypertrophy.48 The altered DNA methylation of H19 in calcified aortic valve disease has recently been proposed to promote mineralization by silencing NOTCH1.49

Here, we found upregulation of H19, but not miR-675, in 2 murine models of AAA, as well as in a preclinical PPE-induced AAA model using Yucatan Ldlr−/− mini-pigs, and in advanced human AAAs. Deletion of miR-675 from H19 did not alter the apoptotic potency or proliferative suppression of H19 in aortic SMCs, nor the overall disease state. This independency from the miR-675 effect may partially account for the cell subtype-specific potency of H19 in cancer cells, human umbilical vein endothelial cells, and aortic SMCs.

An additional important feature, which likely determines the functionality of H19, relates to its specific protein interactions. Several transcription factors, including E2F transcription factor 1 (E2F1),50 p53,51 and c-myc,52 have been reported to interact with H19 and modulate its expression in human cancers. Given the cellular effects of H19 modulation in SMC fate decisions, we speculated that H19 could be operating through transcription factors in aneurysm disease progression as well. Therefore, we selected a panel of transcription factors with predicted binding to H19. HIF1α was the only deregulated transcription factor in response to overexpression and depletion of H19 in hAoSMCs. Functional experiments indicated that HIF1α was essential in mediating the proapoptotic effects of H19. Interestingly, an increase in H19 RNA levels on hypoxia in tumor cells has been reported by Matouk et al,53 and HIF1α was identified as a critical factor for this induction. In our current experiments, we did not observe any significant changes in H19 expression on blocking HIF1α in SMCs. Consistent with our findings, the cited researchers later confirmed that hypoxic induction of H19 was ubiquitous in p53 null cells, and not reversible unless simultaneous suppression of p53 and overexpression of HIF1α in p53 wt cells was performed54. Our previous work has already revealed the importance of increased p53 signaling in experimental AAA development and human disease.29

HIF1α has been implicated in AAA biology before.55 Tissues from human AAA patients express high levels of HIF1α, matrix metalloproteinase (MMP)-2, and Ets-1 within inflammatory infiltrates of the tunica media.55 On exposure to hypoxia, hAoSMCs show enhanced secretion of MMP-2 and MMP-9, contributing to aortic wall weakening and rupture.55,56 HIF1α has furthermore been reported to be coexpressed with vascular endothelial growth factor C and MMP-9 in the intima/media, participating in lymphangiogenesis and angiogenesis in AAAs.57. A population-based study indicated polymorphisms in the HIF1α and vascular endothelial growth factor genes, leading to a potential genetic predisposition for AAAs, independent from peripheral atherosclerosis.58

As the predominant cells in the tunica media of the aorta, SMCs are essential for the maintenance of aortic structure and function. Dysregulation of SMC differentiation and apoptosis largely contributes to the development and progression of AAAs.59,60 Our current discovery that knockdown of HIF1α can abolish H19-induced SMC apoptosis suggests that HIF1α is the main downstream target for H19 and required for the H19-dependent proapoptotic effect on SMCs (Figure 3). Moreover, the expression of HIF1α highly correlated with increased H19 and SMC apoptosis in mouse, Ldlr−/− mini-pig, and human AAA specimens (Figures 4 and 5). Experimental knockdown of H19 resulted in decreased HIF1α expression and reduced AAA growth rates in both murine models (Figure 4, Figure II in the online-only Data Supplement). Our results suggest a promotive role for HIF1α in AAA development, while targeting HIF1α via suppression of H19 exerts protective effects.

In line with this concept, either silencing with lentivirus61,62 or pharmacologically inhibiting63 HIF1α has been shown to reduce aneurysm size via alleviating macrophage infiltration, inhibiting neovascularity, and decreasing MMP2/9 activity in AngII-induced AAAs. It has further been shown that HIF1α-specific deletion in myeloid lineage cells can exaggerate AngII-induced formation of AAAs by augmenting macrophage infiltration and suppressing tissue inhibitors of metalloproteinases (TIMPs).64

HIF1α induction on hypoxia and its relation to SMC apoptosis remains controversial. Overall, it is believed that the severity and duration of hypoxia determines whether cells become apoptotic or adapt to hypoxia and survive.27 Here, we provide evidence that increased HIF1α directly interacts with Mdm2 and prevents Mdm2-mediated reduction of p53, which leads to induced BAX and SMC apoptosis under AAA-relevant (AngII) conditions. Our data reconfirm the importance of p53 in the HIF1α-mediated apoptosis scenario, which has been discussed intensively.27,65

H19 has a highly evolutionary conserved secondary structure, suggesting that the molecular function of H19 is structure-dependent.66 Sequence analysis of H19 indicates several canonical binding sites for the HIF1α transcription factor. Our results illustrate that expression levels of H19 and HIF1α are highly upregulated in AAA tissues from different species and models, as well as in AngII-treated hAoSMCs. Of importance, H19 colocalizes and interacts with HIF1α predominantly in the cytoplasm on stimulation. Furthermore, overexpression of H19 attenuates CoCl2-induced HIF1α translocation into the nucleus. In consequence, H19 facilitates the retention of HIF1α in the cytoplasm, preventing HIF1α translocation into the nucleus, and thereby blocking the activation of genes with prosurvival properties. Intriguingly, such cytoplasmic stabilization of HIF1α via a lncRNA regulatory framework has been reported recently.67 Consistent with a previous study,68 we confirmed that H19 acts in both, cytoplasm and nucleus. In the nucleus, H19 binds to the promoter region of HIF1α. Interestingly, H19 also recruits the transcription factor Sp1, which has been reported as an essential inducer for HIF1α transcription in this region. Our data suggest that within the nucleus, H19 acts as an enhancer, similar to what has been reported for other lncRNAs.39 In the cytoplasm, H19 mainly serves as a scaffold for regulatory proteins68 or miRNAs.45

In conclusion have we identified a lncRNA of functional relevance in AAA development, its progression, and end-stage disease (Figure 8). In the nucleus of SMCs, augmented H19 binds to the promoter region of HIF1α and recruits the transcription factor Sp1, which enhances HIF1α expression. Meanwhile, H19 interacts with HIF1α protein and retains it within the cytoplasm, serving as a scaffold that can block prosurvival signaling. Increased cytoplasmic HIF1α directly interacts with Mdm2 and prevents Mdm2-mediated reduction of p53, which leads to elevated BAX and decreased BCL2 levels. This triggers SMC apoptosis and accelerates AAA development and progression.

Figure 8.

Figure 8. Schematic mechanism of action for H19 and its role in abdominal aortic aneurysm (AAA) development and progression. Our studies indicate that H19 is a novel regulator of smooth muscle cell survival during AAA expansion. In the nucleus, increased levels of H19 bind to the promoter region of HIF1α and recruit the transcription factor Sp1, which enhances HIF1α expression. In the cytoplasm, H19 retains the HIF1α protein, which results in p53 stabilization, serving as a scaffold that can limit prosurvival signaling (indicated by elevated BAX and decreased BCL2 levels). Taken together these events trigger smooth muscle cell apoptosis, aggravating AAA development and disease progression.

Acknowledgments

The authors highly appreciate that Prof Xu Gao and Dr Ning Ma (Harbin Medical University, China) and Prof Eric Adriaenssens (INSERM U908, University of Lille, France) kindly provided H19 wt/mutant vectors. All have provided permission to be named in this article.

Footnotes

Sources of Funding, see page 1566

The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCULATIONAHA.117.032184.

https://www.ahajournals.org/journal.circ

Lars Maegdefessel, MD, PhD, Vascular Biology Unit, Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar, Technical University Munich, Ismaninger Strasse 22, 81675 Munich, Germany. E-mail

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