Cardiovascular Research (2020) 116, 1288–1299 doi:10.1093/cvr/cvz239 High-density lipoprotein remodelled in hypercholesterolaemic blood induce epigenetically driven down-regulation of endothelial HIF-1a expression in a preclinical animal model Soumaya Ben-Aicha 1,2, Rafael Escate 1,3, Laura Casanı́ 1 , Teresa Padró 1,3, ~ a 1,3, Gemma Arderiu 1, Guiomar Mendieta Esther Pen 1,2,4 , Lina Badimón1,3,5†, and Gemma Vilahur 1,3*† 1 Cardiovascular Program—ICCC, Research Institute-Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain; 2School of Medicine, University of Barcelona (UB), Barcelona, Spain; 3Centro de Investigación Biomédica en Red Cardiovascular (CIBERCV), Instituto de Salud Carlos III, Madrid, Spain; 4Cardiology Department, Hospital Clinic Barcelona, Barcelona, Spain; and 5Cardiovascular Research Chair, Universidad Autónoma Barcelona (UAB), Barcelona, Spain Received 3 May 2019; revised 24 July 2019; editorial decision 23 August 2019; accepted 29 August 2019; online publish-ahead-of-print 30 August 2019 Time for primary review: 31 days Aims High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL- transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to re- cipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolae- mia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs). .................................................................................................................................................................................................... Methods Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching and results cholesterol levels of 321.0 (229.7–378.5) mg/dL and 74.0 (62.5–80.2) mg/dL, respectively. HDL particles were iso- lated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were deliv- ered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine admin- istered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7, 1.7, and 1.3, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (-1.6, -1.4, respectively) in lower levels in HC-HDL. miR-126- 5p and -3p were transferred from HC-HDL to EC (2.5; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1a was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1a was diminished in the coronary endothelial layer of NC pigs adminis- tered HC-HDL vs. those administered NC-HDL (P < 0.05). .................................................................................................................................................................................................... Conclusion Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1a. 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 * Corresponding author. Tel: þ34 93 5537100; fax: þ34 935565559, E-mail: gvilahur@santpau.cat † The last two authors contributed equally to the work. C The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology. V This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com HDL induce HIF-1a down-regulation 1289 Graphical Abstract .................................................................................................................................................................................................... Keywords HDL • Dyslipidaemia • Translational research • miRNAs • Endothelial cells .. 1. Introduction .. reverse cholesterol transport and ischaemia/reperfusion injury.3–5 In this .. regard, HDL-mediated transport and delivery of miRNAs to recipient .. MicroRNAs (miRNAs) are short, non-coding RNAs (22 nucleotide in .. cells can regulate expression of genes involved in lipid metabolism, in- length) that control gene expression through post-transcriptional regula- .. flammation, angiogenesis, and apoptosis.6–9 .. tion of target mRNAs. Extracellular miRNAs have been detected in mul- ... Robust epidemiological data supports that increasing HDL-choles- tiple lipid-based carriers including exosomes, microparticles, and .. terol (HDL-c) levels predict improved cardiovascular outcomes; how- lipoproteins.1 Indeed, high-density lipoproteins (HDLs) are lipoproteins .. .. ever, HDL-c raising therapies in secondary prevention trials have failed that in addition to cholesterol, transport other lipid species, proteins, .. to demonstrate cardiovascular benefits.10 As such, within the last de- .. and miRNAs.2 HDL particles exert multiple cardiovascular protective .. cade, it has become increasingly evident that the measure of the choles- functions. Under physiological conditions HDL-cargo molecules that .. terol levels transported by HDL (HDL-c) is not a good read-out of HDL .. maintain cardiovascular homeostasis and function by promoting circulat- .. beneficial effects because the presence of excess cholesterol (hypercho- ing cells, endothelial cells (ECs), vessel wall stabilization, and regulating .. lesterolaemia) increases the transport of cholesterol in detriment of 1290 S. Ben-Aicha et al. other beneficial cargos. Moreover, the presence of comorbidities modi- .. Protein Assay Kit, Thermo Fisher Scientific, Waltham, Massachusetts, .. fies HDL particle composition and function hampering HDL-beneficial .. USA). Lipoprotein cholesterol content was assessed by the Cholesterol effects and even becoming deleterious.11–16 We have recently demon- .. Quantification Kit (MAK043-Sigma-Aldrich; MO, USA). The purity of the .. strated that HDL in vivo remodelling is affected by low-density .. HDL fraction was confirmed by excluding the presence of ApoB (by-aga- lipoprotein (LDL) cholesterol levels. Indeed, the presence of hypercho- .. rose gel electrophoresis), microvesicles (FACS for Anexin Vþ microve- .. lesterolaemia influences HDL lipidomic and proteomic profile and ren- .. sicles), and exosomes (western blots for transpanins CD63 and CD81; ders HDL particles dysfunctional.17–19 Particularly, HDL particles .. .. exosomes isolated by ExoMirTM Kit).23 formed under hypercholesterolaemic conditions, as compared to HDL .. particles formed in a normocholesterolaemic milieu, have lower levels of .. .. 2.1.3 HDL miRNA isolation phosphatidylcholine-lipid species and higher levels of cholesteryl esters .. HDL samples were thawed on ice and centrifuged at 3000 g for 5 min at ..  and a lower cargo of several proteins with known cardioprotective func- . 4 C. An aliquot of 200 lL per sample (protein concentration HC: 5.7 mg/ tions including lipocalin retinol binding protein-4 and apolipoprotein-M. mL and NC: 5.6 mg/mL) was transferred to a FluidX tube and 60 lL Interestingly, we have further proved that such adverse structural of lysissolution buffer containing 1 lg carrier-RNA per 60 lL lysis remodelling is associated with a marked reduction in HDL anti-oxidant solution buffer and RNA spike-in template mixture were added to the and cholesterol efflux capacity and complete loss of HDL-induced cardi- sample and mixed for 1 min and incubated for 7 min at room tempera- oprotection.20 However, to date, there is no information on the impact ture, followed by addition of 20 lL protein precipitation solution buffer. of hypercholesterolaemia on HDL-transported miRNA (HDL-miRNA) Total RNA was extracted from the samples using miRCURY RNA isola- content or on the possible effects of HDL-miRNAs on EC gene regula- tion Kit through a high throughput bead-based protocol v.1 (Exiqon, tion. Accordingly, in the present study, we aimed to investigate the Vedbaek, Denmark) in an automated 96-well format. The purified total HDL-miRNA cargo of HDL formed in vivo in hypercholesterolaemic RNA was eluted in a final volume of 50 lL and its optimal quality conditions and whether the tentative modifications were able to differ- checked. entially regulate gene expression in target cells. 2.1.4 HDL miRNA analysis by qPCR Seven microlitres of RNA were reverse transcribed in 35 lL reactions 2. Methods using the miRCURY LNATM Universal RT microRNA PCR, Experimental procedures were reviewed and approved by the Polyadenylation and cDNA synthesis kit (Exiqon). cDNA was diluted Institutional Animal Care and Use Committees (CEEA-IR) and autho- 50 and assayed in 10 mL PCR reactions according to the protocol for rized by the Animal Experimental Committee of the local government miRCURY LNATM Universal RT microRNA PCR. A panel of 149 (#5601) in accordance to the Spanish law (RD 53/2013) and European miRNAs homologous for pig and human were analysed in HDL Directive 2010/63/EU. In addition, the investigation conforms to the (Supplementary material online, Table S3). Each miRNA was assayed by Guide for the Care and Use of Laboratory Animals published by the US qPCR (microRNA Ready-to-Use PCR, Custom Pick and Mix panel using R National Institutes of Health (NIH Publication No. 85-23, revised 1985), ExiLENT SYBRV Green master mix) and negative controls were run in R follows the ARRIVE guidelines and is committed to the 3Rs of laboratory parallel. The amplification was performed in a LightCyclerV 480 Real- animal research and consequently used the minimal number of animals Time PCR System (Roche) in 384-well plates. The amplification curves to reach statistical significance.21 All animals were allowed to acclimate were analysed using the Roche LC software, both for Cq determination 7 days prior to any intervention and housed in individual cages under (following the 2nd derivative method) and for melting curve analysis. light-controlled conditions and room temperature. 2.1.5 HDL miRNA data analyses 2.1 Experimental model of The amplification efficiency was calculated (LinReg software). All assays hypercholesterolaemia and HDL isolation were inspected for distinct melting curves and the Melt temperature 2.1.1 Experimental animal model (Tm) was checked to be within known specifications for the assay. Four-month-old pigs (N = 20; weight: 40kg) randomly received either Assays had to be detected beyond 5 Cqs of the negative control and regular normocholesterolaemic chow (NC; n = 10) or a high-fat/high- with Cq < 37 in order to be included in the data analyses. The cholesterol diet (HC; n = 10) for 10 days (composition of the diet is de- NormFinder helped to find out the best possible normalizer which tailed in Supplementary material online, Table S1). HC animals developed resulted to be the average of the best assays detected in all samples high LDL-cholesterol levels similar to those found in hypercholesterolae- (n = 20). The formula used to calculate the normalized Cq values was mic humans, 321.0 (229.7–378.5) mg/dL, while NC-animals had 74.0 the following: Normalized Cq = average Cq (n = 20) - assay Cq (sample). (62.5–80.2) mg/dL (P < 0.0001; Supplementary material online, Table S2).22 We then performed a comparative analysis between NC and HC iso- lated HDL-miRNAs. Data were expressed as fold-change. 2.1.2 HDL isolation and purification On Day 10, animals were tranquilized by intramuscular injection of 2.2 Cell culture studies Azoperone (40 mg/mL; 7 mg/kg), anaesthesia was induced by propofol 2.2.1 Porcine aortic endothelial cells isolation, expansion (2%; 3 mg/kg), and maintained by isofluorane inhalation (3%). Animals and treatment were then sacrificed by propofol overdose and potassium chloride Aortic ECs were obtained from regular-fed pigs by collagenase digestion (2 M). HDLs were isolated by standard sequential preparative ultracen- as previously described.24 Cells were cultured in growth M199 (Gibco) trifugation techniques from plasma-EDTA (ethylenediaminetetra-acetic medium supplemented with 10% FBS and antibiotics (0.1 mg/mL strepto- acid) before sacrifice as previously reported.19 Protein levels in HDL mycin, 100 U/mL penicillin) until confluence. Cells were detached by were quantified by standard Bradford protein assay (PierceTM BCA trypsin/EDTA treatment and seeded into larger flasks for expansion. HDL induce HIF-1a down-regulation 1291 .. Forty-eight hours after plating 300 000 cells in six well plates, 0.1% FBS .. samples. The amplification was performed in ABI PRISM 7900HT was added to the medium for 24 h and then EC were incubated with .. Sequence Detection System (Applied Biosystems; CA, EU) in 384-well .. NC-HDL (50 mg/mL protein), HC-HDL (50 mg/mL protein), or PBS/con- .. plates. The amplification curves were analysed using the Applied trol for 18 h (in duplicates; n = 5) as previously reported.24,25 EC viability .. Biosystems Sequence Detection System 2.4.1 software. Ct values were .. was determined by trypan blue exclusion. ECs were always used be- .. obtained and processed by the standard curve method and normalized tween the third and seventh passage. .. with the endogenous gene. .. .. 2.2.2 ECs and plasma miRNA isolation and data analysis .. 2.3.3 Protein expression validation .. ECs were collected and total RNA was obtained with mirVANA miRNA .. Cell lysis was performed according to the previous established proto- .. col.28 The cell protein lysates were stored at -80 C until use. Protein R kit. The RNA was transcribed using TaqManV Advanced miRNA cDNA .. Synthesis Kit (Applied Biosystems; CA, EU) protocol. All assays were .. concentrations were assessed using the BSA assay and then SDS– .. performed in duplicates for the differentially expressed miRNAs includ- .. polyacrylamide gel electrophoresis (SDS-PAGE) was run. Blots were ing miR-126-5p and -3p, let7g-5p, miR-103a-3p and miR-30b-5p (Life .. blocked with BSA at room temperature for 1 h and incubated with the .. Technologies; CA, EU) (n = 5). For normalization miR-423-3p and let- .. appropriate primary antibody [HIF1a (sc-10790, Santa Cruz 7a-5p means were used as endogenous miRNA controls.26 The amplifi- .. Biotechnology, Dallas, TX, USA)] at 4 C overnight. Membranes were .. cation was performed in ABI PRISM 7900HT Sequence Detection .. then incubated with a horseradish peroxidase-conjugated anti-mouse System (Applied Biosystems; CA, EU) in 384-well plates. The amplifica- .. antibody for 2 h. Densitometry analyses were performed using commer- .. tion curves were analysed using the Applied Biosystems Sequence .. cially available quantitative software (ImageLab Software, Bio-Rad Detection System 2.4.1 software. Ct values were obtained and processed .. Laboratories, CA, USA) with the control representing 1.0-fold. .. by the standard curve method and normalized with the endogen. To ob- .. tain fold difference vs. control the ratio sample/control was calculated. .. .. 2.3.4 Mir126 target validation by miR126-mimic and Networks, functional analyses, detection of miRNAs targets and interac- .. miR126 inhibitor .. tions were generated by Ingenuity Pathway Analysis software (IPA) and .. ECs were transfected with miR126-mimic or miR126 inhibitor (both for RNA hybrid tool. .. -5p and -3p) (Life Technologies; CA, EU) with the LipofectamineTM .. .. RNAiMAX Transfection Reagent (Life Technologies; CA, EU) following 2.2.3 Assessment of HDL-miRNA transfer to ECs .. the supplier recommendations. Then, ECs were treated with HDL-HC .. To investigate whether the scavenger receptor class B member 1 (SRB1) .. or HDL-NC as reported above. A miRNA Mimic Negative Control #1 was involved in HDL-miRNA transfer to ECs a specific SRB1 blocker, .. .. (Life Technologies; CA, EU) was run in parallel. BLT1 (EMD Millipore Corp, USA; 8 mM), was added to ECs prior to .. .. HDL incubation.25 In addition, and taking into consideration that miR- .. 2.4 In vivo validation of miR-126 target 126 (both 5p and 3p) is encoded in the 7th intron of the EGFL7 gene, we .. genes by confocal microscopy .. assessed the expression of both EFGL7 and the pre-miRNA sequence .. We assessed HIF1a protein expression in the coronary arteries of nor- (pre-mir-126; n = 5) in ECs in order to ascertain whether the potential .. mocholesterolaemic pigs (n = 12) administered HC-HDL (n = 6) or NC- .. intracellular changes detected in miR-126-5p and -3p expression were .. HDL (n = 6). In this regard, we processed the coronary arteries from related to an up-regulation of its cellular synthesis or because of transfer .. .. pigs of our previous published study19 which had been kept in our tissue from HDL. Pri-miR126 could not be investigated because there is no .. biobank at -80 C until use. Accordingly, we fixed the proximal portion commercial assay available for Sus scrofa so far. .. .. of the coronary artery of all pigs in 4% paraformaldehyde and the .. paraffin-embedded for confocal microscopy analysis. Serial sections 2.3 miR126 target gene analysis .. .. (5 mm thick) were incubated with blocking buffer and then with antibody 2.3.1 In silico analysis .. against HIF1a (H1alpha67, Novus Biologicals, Littleton, CO, USA). .. Potential target genes were obtained by using databases TargetScan, .. Preparations were counterstained with fluorescently labelled secondary miRwalk (5UTR, CDS, 3UTR) and the Ingenuity Pathway Analysis (IPA; .. antibody (Invitrogen) and mounted with fluorescent medium. .. http://www.ingenuity.com/), according to their search algorithms. The .. Fluorescent images were acquired in a scan format of 1024  1024 pix- functionality of target genes in cellular processes was predicted by IPA .. els in a spatial data set (x y z). Controls with no primary antibody showed .. Core analysis. Furthermore, RNAhybrid (Bielefeld University .. no fluorescence labelling. Bioinformatics server) was used for calculating minimum free energy .. .. (MFE), which indicates the accessibility of miRNAs at the target gene .. 2.5 Statistics binding sites. Target genes were selected by comparison of MFE .. .. The Shapiro–Wilk test was employed to analyse data normal or non- values.27 .. normal distribution; data are reported as the mean ± standard deviation .. .. and median (interquartile range), respectively. For the biochemical de- 2.3.2 Gene expression validation .. scriptive data and SRB1 in vivo analysis, we performed non-parametric .. RNA was transcribed using the High Capacity cDNA Archive kit .. test Kruskal–Wallis with Dunn’s multiple comparisons corrected test. (Applied Biosystems, Foster City, CA, USA) protocol for genomic RNA. .. Tukey’s honest significance corrected test was used in conjunction with .. qPCRs were performed in duplicates for SCARB1, mir-126-5p and -3p, .. an ANOVA (post hoc analysis) for multiple comparisons to find signifi- HIF1a, vascular cell adhesion protein 1 (VCAM1), vascular endothelial .. cantly difference between group means due to its condition of indepen- .. growth factor A (VEGFA) (Life Technologies; CA, EU), and EGFL7 (Bio- .. dent samples. Accordingly, t-test was used for two group independent Rad Laboratories, CA, EU). 18SrRNA was used as endogenous control .. samples mean comparison. A two-sided P-value less than 0.05 was con- .. for tissue samples and GAPDH (Life Technologies; CA, EU) for EC . sidered statistically significant. 1292 S. Ben-Aicha et al. Figure 1 HeatMap including the miRNAs detected in all HDL samples isolated from hypercholesterolaemic (HC; n = 10) and normocholesterolaemic (NC; n = 10) pigs. Sample size: For the EC approach accepting a risk of 0.05 and b risk of .. whereas, conversely, the amounts of miR103a-3p and let7g-5p were sig- .. 0.2 in a two-sided test, an n = 3 was necessary to recognize as statistically .. nificantly lower (-1.6, -1.4, respectively). significant difference >_0.5 units. Yet, we performed n = 5 in all conditions .. .. to make our conclusions more robust. The common standard deviation .. is assumed to be 0.2 and it we anticipated a dropout rate of 0% .. 3.3 HC-HDL particles transfer miR-126 to .. (GRANMO software, Spain). All statistical analyses and graphics were per- .. ECs through a SRB1-dependent .. formed using GraphPad Prism 7.0 version software. .. mechanism .. ECs incubated with HC-HDL showed significantly higher intracellular .. .. levels of miR-126-5p (P < 0.0001) and miR126-3p (P < 0.0001), while lev- 3. Results .. els of both miR-126s (5p and 3p) were similar in control ECs and ECs in- .. .. cubated with NC-HDL (Figure 2A and B). No differences were observed 3.1 HDL-characterization .. Cholesterol content was two-fold higher in HC- than in NC-HDLs .. for miR103a-3p (Figure 2C), miR30b-5p (Figure 2D), and let7g-5p .. (Figure 2E). (Supplementary material online, Table S4). This observation is in line .. with our previous findings regarding the effects of hypercholesterolaemia .. Interestingly, the internalization of miRNA-126 in ECs was mediated .. by SRB1.We found that SRB1 blockade by BLT-1 abolished HC-HDL- on modifying HDL-lipid composition.20 The isolated HDL samples were .. .. induced increase in miR-126 (both 5p and 3p) (Figure 3A and B) content. free from ApoB (Supplementary material online, Figure S1A), Anexin Vþ .. SRB1 expression in ECs did not differ by incubation with either NC- or microvesicles (Supplementary material online, Figure S1B), and exosomes .. .. HC-HDLs (Supplementary material online, Figure S2A). Moreover, com- (Supplementary material online, Figure S1C), supporting the purity of the .. HDL preparations. The protein concentration was similar in HC-HDL .. parable SRB1 transcript levels were detected in pig coronary arteries .. and liver (Supplementary material online, Figure S2B). Therefore, SRB1 (5.7 mg/mL) and in NC-HDL (5.6 mg/mL). .. .. was found abundantly expressed in pig ECs and its expression was not .. influenced by incubation with HDL. Finally, blockade of SRB1 in HDL- 3.2 HDL-related miRNA profile .. Among the 149 miRNAs analysed, 30 miRNAs were detected in all HDL .. and PBS-incubated cells led to comparable up-regulation in SRB1 levels .. samples (Figure 1 and Supplementary material online, Table S5). Among .. (Supplementary material online, Figure S3). these 30 miRNA, the amounts of five miRNAs were significantly different .. To further confirm the transfer of miR-126-5p and -3p from HC-HDL .. in HC- and in NC-HDL particles (P < 0.05). Particularly, there was a .. to the ECs, we analysed pre-mir-126 and EGFL7 levels. As shown in higher content of miR126-5p, miR126-3p, and miR-30b-5p in HC-HDL .. Figure 3, both pre-mir-126 (Figure 3C) and EGFL7 (Figure 3D) were simi- .. particles as compared to NC-HDL (2.7, 1.7, and 1.3, respectively), . larly expressed among all the three groups excluding an enhanced HDL induce HIF-1a down-regulation 1293 Figure 2 Endothelial cells incubated with hypercholesterolaemic-HDL show higher miR-126-5p and 3p levels as compared to normocholesterolaemic- HDL [miR126-5p (A) and miR126-3p (B)], whereas no differences were observed for 103a-3p (C), miR-30b-5p (D), or let-7g-5p (E) levels. n = 5. Data are reported as mean ± SD. *ANOVA test P < 0.05 vs. Control and NC-HDL groups. HC: hypercholesterolaemic; NC: normocholesterolaemic. .. intracellular transcription of miRNA-126 in HC-HDL-treated cells and .. To further validate HIF1a as a target gene for miR126, we examined confirming transfer from HC-HDL. .. the effects of miR-126-mimics and miR-126-inhibitors (both -5p and -3p) .. .. in ECs (Figure 4). Indeed, control ECs transfected with both miR126- .. mimics showed lower HIF1a transcript levels and protein expression 3.4 miR-126 target genes on ECs .. We used different computational databases (Supplementary material on- .. than ECs transfected with the negative control and both miR126- .. inhibitors (Figure 4A–D and Supplementary material online, Figure S5). line, Figure S4 and Table S6) to identify potential target genes of miR-126- .. .. HIF1a gene expression was lower in negative-control transfected ECs 5p and -3p in ECs, and only those identified in at least two databases .. incubated with HC-HDLs as compared to NC-HDL and control were further investigated. We identified a total of 7597 candidate target .. .. (P < 0.05; Figure 4E and F and Supplementary material online, Figure S5). genes potentially regulated by miR-126 (3p-strands: 454; 5p-strands: .. In the EC cultures treated with HDL the effects of HL-HDL on the 7143). Among them, common target genes were identified from at least .. .. down-regulation of HIF1a expression were abrogated by miR126-5p/3p two databases (51 for miR-126-3p and 942 for miR-126-5p). HIF1a was .. inhibitors (Supplementary material online, Figure S6). .. the potential best target gene for miR-126-5p regulation given the low .. Based on these findings, we proceeded to validate HIF1a by qPCR MFE values (-8.3, -11.2, -12.6, -12.1, -8.5, -13.1 Kcal/mol) and its six tenta- .. (n = 5) and found that HIF1a transcript levels were significantly down- .. tive binding sites for miR126-5p (Table 1). The best potential target for .. regulated in ECs treated with HC-HDL (P < 0.01 vs. PBS/control; miR126-3p was DR5 but it could not be validated because its sequence .. P < 0.006 vs. NC-HDL; Figure 5A). HIF1a protein levels were also signifi- .. is not yet available in S. Scrofa. . cantly reduced in HC-HDL treated ECs (Figure5B). 1294 S. Ben-Aicha et al. Figure 3 Hypercholesterolaemic HDL particles transfer miR-126-5p and -3p (A and B, respectively) to endothelial cells through a SRB1-dependent mech- anism. No differences are detected for Pre-mir-126 (C) or for miR-126 encoding gene EGFL7 (D) levels among the different groups. n = 5. Data are reported as mean ± SD. *ANOVA test P < 0.05 vs. any other group. HC: hypercholesterolaemic; NC: normocholesterolaemic. .. Mimic-MiR126 (-5p and -3p) induced down-regulation of VCAM1 .. -3p content is higher in HC-HDL particles; (iii) HC-HDL transfer miR- mRNA expression (Supplementary material online, Figure S7A and B) .. 126 to ECs through a SRB1-dependent mechanism; (iv) internalized .. and up-regulation of VEGFA mRNA expression (Supplementary mate- .. miRNA-126 regulates HIF1a in recipient cells. HIF1a down-regulation is rial online, Figure S7C and D). .. observed in vivo as seen in the coronary arteries of animals treated with .. .. HL-HDL but not in animals treated with NL-HDL. .. 3.5 In vivo validation of HC-HDL-related .. During the last years, it has become increasingly evident that HDL can .. transport multiple miRNAs including miR-12629, miR-10330, let-7g, miR- miR-126 potential to modulate endothelial .. .. 222, and miR-2236 among others. However, it remained to be deter- HIF1a .. .. mined whether the presence of cardiovascular risk factors altered the Finally, to determine whether our in vitro findings translated into the .. HDL transported miRNA signature. In the present study, we demon- in vivo setting, we assessed HIF1a expression by confocal microscopy in .. .. strate that HDL formed under hypercholesterolaemic conditions, be- the coronary arteries of NC pigs administered HC- or NC- HDLs. .. cause of high dietary fat intake, show a higher abundance of the miRNAs Interestingly, as shown in Figure 6, expression of HIF1a was markedly re- .. duced in the intimal layer of the coronary arteries of those NC animals .. miR-126-5p and -3p, and miR-30b-5p and, conversely, a lower amount .. of miRNAs miR-103a-3p and let-7g-5p as compared to those HDL administered HC-HDLs as compared to those administered NC-HDLs. .. .. formed in a normocholesterolaemic milieu. Interestingly, Vickers et al.6 .. had reported a differential HDL-miRNA signature (particularly miR223 .. .. levels) in familial hypercholesterolaemia patients that have high LDL- 4. Discussion .. .. cholesterol levels during life, due to the inherited condition of the dis- In the present study, we report that: (i) HDL formed in hypercholestero- .. ease. So far, a positive association between miR-126 levels in plasma and .. laemic conditions transport different amounts of miRNAs than those .. in HDL has been described, as well as in coronary artery disease and HDL formed in normocholesterolaemic conditions; (ii) miR-126-5p and .. myocardial infarction.29,31,32 Furthermore, higher amount of miR-126 HDL induce HIF-1a down-regulation 1295 Figure 4 Control ECs transfected with miR126-mimic show lower HIF1a gene levels as compared to ECs transfected with the negative control both at mRNA expression and protein level (A–D). HIF1a-mRNA expression was lower in negative-control transfected ECs incubated with HC- as compared to NC-HDLs and control (E and F). *ANOVA test P < 0.05. HC: hypercholesterolaemic; NC: normocholesterolaemic. .. was found in HDL particles of overweight and obese subjects,33,34 and .. LDL-cholesterol levels carry a high content of miR-126, which is deliv- we have recently shown that miR-126 regulates monocyte/macrophage .. ered into ECs via a SRB1, leading to the down-regulation of its target .. differentiation under a rich LDL-cholesterol environment.28 Indeed, a .. gene HIF-1a. As such, SRB1 blockade inhibited miR-126 (both -5p and - positive association between miR-126 expression and LDL-cholesterol .. 3p) delivery into ECs pointing to the critical role of this receptor in .. levels has been demonstrated.35 Here, we demonstrate that HDL par- .. HDL-miRNA internalization into target recipient cells. This finding is fur- ticles formed in a diet-induced hypercholesterolaemic milieu with high .. ther supported by the fact that no changes are detected in pre-mir-126 1296 S. Ben-Aicha et al. Figure 5 mRNA expression and protein levels of miR126 target HIF1a in endothelial cells. n = 5. Data are reported as mean ± SD. *ANOVA test P < 0.05 vs. Control and NC-HDL groups. HC: hypercholesterolaemic; NC: normocholesterolaemic. (mir-126) or its encoding gene EGFL7.36 SRB1 is a membrane receptor .. angiogenesis and blood vessel integrity by directly repressing the .. involved in the selective uptake of cholesteryl ester from HDL. SRB1 is .. Sprouty Related EVH1 Domain Containing 1 (SPRED1)43 gene and the highly expressed in the liver and other cells including monocytes/ .. phosphoinositide-3-kinase regulatory subunit 244,45 with the consequent .. macrophages, ECs, smooth muscle cells, adipocytes, and steroidogenic .. increase in VEGFA.46 Moreover, miR-126 has also shown to reduce .. cells.37–39 The ability of HDL particles to transfer cargo-miRNAs into re- .. leucocyte adherence and vascular inflammation by targeting VCAM-1.47 cipient cells via SRB1 had already been described in macrophages and .. On the other hand, LDL-cholesterol and miRNA-126 levels are posi- .. hepatocytes.6 In fact, Vickers et al.6 reported that SRB1 over expression .. tively associated in patients with asymptomatic coronary artery disease associated with the greatest cellular uptake of HDL-miRNAs. .. and have been suggested to exert prognostic value for future cardiovas- .. Whereas in rodents SRB1 is expressed most abundantly in tissues .. cular events.48 In line with previous work, we further confirm the ability that efficiently utilize cholesterol ester (biliary cholesterol secretion or .. of miR-126 to down-regulate endothelial VCAM-1 expression and indi- .. steroid hormone production),40 in humans SRB1 is homogenously dis- .. rectly enhance VEGFA transcript levels; yet, we further demonstrate the tributed in all cells.41 In the present study, we show that in pigs there are .. ability of both miR-126s’ (5p and 3p) to reduce endothelial HIF1a gene .. comparable SRB1 expression levels in the liver and arterial beds. It is .. levels. Moreover, our in vitro findings are further recapitulated in vivo .. possible that SRB1 serves as docking receptor for HDL to transfer their .. where HIF1a protein levels are lower in the intima of the coronary ar- miRNA-cargo into target cells. Although the SRB1 is the endothelial re- .. teries of hypercholesterolaemic pigs. Although the complexity of the .. ceptor mainly involved in HDL-miRNA transfer, we cannot exclude that .. in vivo model does not allow to exclude the existence of other potential other HDL receptors (ABCA1, ABCG1) may also be involved in HDL- .. mechanisms modulating HIF-1a, all animals were kept under identical .. miR126 transfer to ECs.42 .. conditions except for the type of infused HDL. It is, therefore, plausible The role of miR-126 on the cardiovascular system is controversial, .. to consider that the changes observed in endothelial HIF1a may derive .. with opposed results in different studies. miR-126 has shown to regulate . from HC-HDL-miR126 transfer. HIF1a is a transcription factor that HDL induce HIF-1a down-regulation 1297 Figure 6 Confocal microscopy analysis of HIF-1a detection in the endothelial layer of the coronary artery of normocholesterolaemic (NC) pigs adminis- tered NC- or hypercholesterolaemic (HC)-HDLs. Representative images of HIF1a (green) detection (A) and bar-graph quantification (B). Data are reported as mean ± SD. n = 6 animals/group. *T-test P < 0.05 vs. NC pigs administered HC-HDL. serves as a hypoxia sensor to induce the expression of angiogenic genes. .. in miR-126 that can be delivered to ECs interfering with key vascular .. Yet, under normoxic conditions, HIF1a is known to modulate compen- .. functions. Collectively, our data provides further insights as to the impact satory and adaptive mechanisms. In this regard, Liu et al.49 have recently .. of hypercholesterolaemia on HDL biogenesis, HDL biology, and HDL .. described in cancer cells that suppression of miR-126 increases HIF1a .. composition as well as on the potential outcomes on HDL-miRNA .. expression promoting metastasis and therapeutic resistance. We evi- .. intercellular communication. Whether miR-126 may serve as a potential dence, in ECs, that overexpression of miR-126 reduces HIF1a expres- .. therapeutic target or whereas reversing hypercholesterolaemia (statin .. sion likely interfering with metabolic adaptation, innate immune .. treatment, diet approaches) may restore HDL-related miRNA normal response, survival and apoptosis.50 On the other hand, HIF1a has been .. profile deserve to be investigated. .. described to be modulated, under hypoxic conditions, by miR-155,51 .. miR-18a, miR-199a, miR-429, miR-433,52 and miR-497195.53,54 The .. .. exact mechanism by which HDL are loaded with miR-126 under hyper- .. Supplementary material cholesterolaemic condition is not known. Biophysical studies have sug- .. .. Supplementary material is available at Cardiovascular Research online. gested that dipolar lipids facilitate small RNAs incorporation within HDL .. particles.55 In this regard, we could speculate that the reported changes .. Conflict of interest: none declared. .. in lipid composition observed in HC-HDL particles20 may have favoured .. miR-126 association to circulating HDLs. Yet, further studies are needed .. .. to confirm this hypothesis and determine the relevance of modulating .. Acknowledgements miR-126 expression in the vascular wall. In this regard, the in vivo func- .. We gratefully acknowledge the valuable help and support of M.A. .. tional impact of miR-126/HIF1a deserves to be investigated. .. Canovas, P. Catalina, O.Garcı́a, J. Moreno, L. Nasarre, F.J. Rodriguez, and In conclusion, we demonstrate that diet-induced hypercholesterolae- .. M. Pescador with animal handling and for the proper conduct of all the .. mia leads to alterations in HDL-miRNA profile favouring an enrichment . experimental and molecular work. 1298 S. Ben-Aicha et al. .. .. Funding ............................................................................................................................................................................................................................................................................................................ .. This work was supported by the Plan Nacional de Salud (PNS) [PGC2018- 212.6 213.1 .......................................................................................................................................................................................................................................................................................................... -12.5 The potential target genes for miR-126-5p related with the top 10 cellular functions by core analysis, a tool from IPA (Ingenuity Pathway Analysis). The cellular functions are showed by P-values according to score. Minimum Free Energy (MFE) .. .. 094025-B-I00 to G.V and SAF2016-76819-R to L.B.] from the Spanish .. Ministry of Science and Innovation and funds FEDER ‘Una Manera de Hacer .. 211.2 28.5 -13.3 -9.6 -14 .. Europa’; a grant from the Spanish Society of Cardiology (Beca FEC MFE values .. Investigación Básica/2016 to G.V); the Instituto de Salud Carlos III (ISCIII) .. [P17/01321 to G.A]; and CIBERCV (to L.B). We thank the support of the .. 28.3 212.1 -12.5 -12.6 -13.1 -17.9 -10.9 -9.4 -8.9 -9.7 -11 .. Generalitat of Catalunya (Secretaria d’Universitats i Recerca del .. Departament d’Economia i Coneixement de la Generalitat, 2017 SGR 1480) .. .. and the Fundación Investigación Cardiovascular-Fundación Jesus Serra for score Total .. their continuous support. 40 29 23 17 16 14 14 12 12 12 .. .. Cell-to-cell signalling .. .. References .. 1. He L, Hannon GJ. Erratum: microRNAs: small RNAs with a big role in gene regula- and interaction .. .. tion. Nat Rev Genet 2004;5:522–531. .. 2. Kontush A, Lindahl M, Lhomme M, Calabresi L, Chapman MJ, Davidson WS. .. 0.00197 Structure of HDL: particle subclasses and molecular components. In Barrett, JE. (ed.). .. .. 3. Handbook of Experimental Pharmacology. Switzerland: Springer Nature; 2015. p. 3–51. Badimon L, Vilahur G. 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Binding of phosphorothioate oligonucleotides to zwitterionic lipo- atic carotid artery disease. Genet Test Mol Biomarkers 2016;20:125–129. . somes. Biochim Biophys Acta 2002;1563:45–52. Translational perspectives Clinical trials aimed at increasing high-density lipoprotein (HDL)-cholesterol in secondary prevention have resulted disappointing. Furthermore, stud- ies have reported on the negative impact of cardiovascular disease on HDL composition and function. The structural/functional changes that suffer HDL particles under cardiovascular comorbid pathological conditions need to be investigated because they render them dysfunctional and even dele- terious. We evidence that diet-induced hypercholesterolaemia (the most prevalent cardiovascular risk factor) alters HDL miRNA signature and these altered miRNAs directly affect vascular homeostasis, adversely regulating the vascular response. Identifying the remodelling changes affecting HDL particles due to pathological conditions will open new avenues for therapeutic intervention.