nutrients Article A Functional Virgin Olive Oil Enriched with Olive Oil and Thyme Phenolic Compounds Improves the Expression of Cholesterol Efflux-Related Genes: A Randomized, Crossover, Controlled Trial Marta Farràs 1,2 , Sara Arranz 3 , Sílvia Carrión 3 , Isaac Subirana 4,5 , Daniel Muñoz-Aguayo 3,6 , Gemma Blanchart 3 , Marjon Kool 3 , Rosa Solà 7,8,9 , María José Motilva 10 , Joan Carles Escolà-Gil 1,2 , Laura Rubió 11 , Sara Fernández-Castillejo 7,8 , Anna Pedret 7,8 , Ramón Estruch 6,12,13 , María Isabel Covas 6,14 , Montserrat Fitó 3,6 , Álvaro Hernáez 6,13, * and Olga Castañer 3,6, * 1 Molecular Bases of Cardiovascular Risk Group, IIB-Sant Pau, 08041 Barcelona, Spain 2 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, 28029 Madrid, Spain 3 Cardiovascular Risk and Nutrition Research Group, Hospital del Mar Medical Research Institute (IMIM), 08003 Barcelona, Spain 4 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain 5 Cardiovascular Genetics and Epidemiology Research Group, IMIM, 08003 Barcelona, Spain 6 CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), ISCIII, 28029 Madrid, Spain 7 Functional Nutrition, Oxidation, and Cardiovascular Diseases Group, Universitat Rovira i Virgili, 43201 Reus, Spain 8 Institut d’Investigació Sanitària Pere Virgili, 43204 Reus, Spain 9 Hospital Universitari Sant Joan de Reus, 43204 Reus, Spain 10 Instituto de Ciencias de la Vid y el Vino-ICVV (Consejo Superior de Investigaciones Científicas-CSIC, Gobierno de La Rioja, Universidad de la Rioja), 26007 Logroño, Spain 11 Food Technology Department, Agrotecnio Center, University of Lleida, 25198 Lleida, Spain 12 Internal Medicine Service, Hospital Clínic de Barcelona, 08036 Barcelona, Spain 13 Cardiovascular Risk, Nutrition and Aging Research Unit, August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain 14 NUPROAS Handelsbolag, Nackă, Sweden; NUPROAS HB, Apartado de Correos 93, 17242 Girona, Spain * Correspondence: alvaro.hernaez1@gmail.com (Á.H.); ocastaner@imim.es (O.C.); Tel.: +34-67-938-4179 (Á.H.); +34-933-160-724 (O.C.)  Received: 11 July 2019; Accepted: 25 July 2019; Published: 26 July 2019  Abstract: The consumption of antioxidant-rich foods such as virgin olive oil (VOO) promotes high-density lipoprotein (HDL) anti-atherogenic capacities. Intake of functional VOOs (enriched with olive/thyme phenolic compounds (PCs)) also improves HDL functions, but the gene expression changes behind these benefits are not fully understood. Our aim was to determine whether these functional VOOs could enhance the expression of cholesterol efflux-related genes. In a randomized, double-blind, crossover, controlled trial, 22 hypercholesterolemic subjects ingested for three weeks 25 mL/day of: (1) a functional VOO enriched with olive oil PCs (500 mg/kg); (2) a functional VOO enriched with olive oil (250 mg/kg) and thyme PCs (250 mg/kg; FVOOT), and; (3) a natural VOO (olive oil PCs: 80 mg/kg, control intervention). We assessed whether these interventions improved the expression of cholesterol efflux-related genes in peripheral blood mononuclear cells by quantitative reverse-transcription polymerase chain reactions. The FVOOT intervention upregulated the expression of CYP27A1 (p = 0.041 and p = 0.053, versus baseline and the control intervention, respectively), CAV1 (p = 0.070, versus the control intervention), and LXRβ, RXRα, and PPARβ/δ (p = 0.005, p = 0.005, and p = 0.038, respectively, relative to the baseline). The consumption of a Nutrients 2019, 11, 1732; doi:10.3390/nu11081732 www.mdpi.com/journal/nutrients Nutrients 2019, 11, 1732 2 of 10 functional VOO enriched with olive oil and thyme PCs enhanced the expression of key cholesterol efflux regulators, such as CYP27A1 and nuclear receptor-related genes. Keywords: functional virgin olive oil; olive oil phenolic compounds; thyme phenolic compounds; cholesterol efflux; HDL cholesterol; transcriptomics 1. Introduction Virgin olive oil (VOO) consumption protects against the development of cardiovascular diseases due to its richness in phenolic compounds (PCs) and other bioactive compounds such as monounsaturated fatty acids [1–4]. Among its beneficial mechanisms, the intake of olive oil PCs has been shown to promote major high-density lipoprotein (HDL) function, particularly cholesterol efflux capacity (HDL’s ability to pick up cholesterol from macrophages), as well as other improvements in HDL quality characteristics [5]. Going one step further, the consumption of enriched or functional VOOs, supplemented with olive oil PCs or other complementary PCs such as those from thyme, has also been shown to improve HDL functional traits and lipid profile [6–10], as well as other molecular mechanisms potentially involved in the development of cardiovascular diseases [11,12]. On the one hand, olive oil PCs are essentially monophenols such as hydroxytyrosol and tyrosol, whose effect on cardiovascular protection [13] (mainly based on their capacity to neutralize pro-oxidant substances) may be complemented with that from more complex PCs such as flavonoids (known to also be able to directly modulate pro-inflammatory pathways such as those involving phosphoinositol-3-kinase/protein kinase B, protein kinase C, and mitogen-activated protein kinases [14]). Beyond the antioxidant and anti-inflammatory effect attributed to olive oil PCs [15,16], their capacity to modulate gene expression is also essential to explain their health effects and those of the dietary patterns in which they are present, such as the Mediterranean diet [17–19]. The benefits of this healthy dietary pattern could be partially attributed to the protective capacities of VOO bioactive compounds on atherosclerosis and neoplastic processes [20]. On the other hand, thyme bioactive compounds such as PCs and essential oils have also been shown to be able to promote HDL cholesterol levels and to decrease low-density lipoprotein cholesterol concentrations in diverse research models [21]. In relation to HDL functionality, the acute consumption of a VOO enriched with its own PCs improved the expression of cholesterol efflux-related genes in pre-/hypertensive subjects in a previous trial [22]. However, the effects of the sustained consumption of functional VOOs on the gene expression related to cholesterol efflux and cholesterol metabolism regulation pathways have not been assessed to date. Thus, the aim of the present study was to assess whether a sustained consumption of functional VOOs (rich in complementary PCs from VOO or thyme) also promoted the expression of this gene pathway, in order to contribute to explaining their potential benefits on HDL function and lipid profile. 2. Materials and Methods 2.1. Olive Oil Preparation Two phenol-enriched VOOs (final PC concentration: 500 ppm) were prepared using as matrix a low-phenolic-content VOO (80 ppm), which also served as the control. In particular, the characteristics of the functional oils were: (1) a functional VOO enriched with an extra quantity of olive oil PCs (500 ppm) (FVOO); and (2) a functional VOO enriched with olive oil (250 ppm) and thyme PCs (250 ppm) (final PC concentration: 500 ppm) (FVOOT). Thyme PCs came from an aqueous thyme extract, not from an essential oil. The procedure to obtain the phenolic extracts and the enriched oils has been previously described [23]. Nutrients 2019, 11, 1732 3 of 10 2.2. Study Design and Biological Samples Thirty-three hypercholesterolemic volunteers (total cholesterol > 200 mg/dL) participated in a randomized, double-blind, crossover, controlled trial in which we compared the effect of the three oils previously described (FVOO, FVOOT, and the control VOO). The intervention consisted of three-week periods in which the volunteers consumed 25 mL/day of raw VOO distributed along meals, preceded by two-week wash-out periods with an olive oil very poor in PCs. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Research and Ethics Committees and registered with the International Standard Randomized Controlled Trial Number ISRCTN77500181 [6]. In the course of the trial, we collected blood samples at fasting before and after each treatment, in order to isolate peripheral blood mononuclear cells (which were conserved at −80 ◦ C for further analysis). We also collected EDTA plasma samples to determine glucose, total cholesterol, and triglyceride concentrations using standard enzymatic automated methods in an ABX Pentra-400 autoanalyzer (ABX-Horiba Diagnostics, Montpellier, France), HDL cholesterol levels by an accelerator selective detergent method (ABX-Horiba Diagnostics), and computed LDL cholesterol by the Friedewald equation whenever triglycerides were <300 mg/dL. In this work, a sub-sample of 22 volunteers was analyzed on the basis of sample availability and quality. Further details of the clinical trial and the preparation of the functional oils have been thoroughly described in previous publications [6]. 2.3. Gene Expression Analyses First, we isolated total RNA from peripheral blood mononuclear cells from blood samples collected from volunteers before and after the three VOO interventions by RNAeasy microkits (Qiagen, Hilden, Germany), quantified total RNA, and checked RNA purity and integrity in a Nanoquant Bioanalyzer (Tecan Ltd., Männedorf, Switzerland). We then converted RNA into complementary DNA using High Capacity cDNA Reverse Transcription kits (Applied Biosystems-Life Technologies, Carlsbad, CA, USA). Finally, we quantified gene expression by the quantitative retrotranscriptase real-time polymerase chain reaction (qRT-PCR) technique, using TaqMan Low Density microfluidic cards (Applied Biosystems-Life Technologies). We selected our candidate genes based on their relationship with cholesterol efflux (cholesterol transporters and efflux transcriptional regulators). We analyzed the data using the Sequence Detection System software 2.4 (Applied Biosystems-Life Technologies) and calculated the changes in gene expression by the relative quantification method (applying the 2−∆∆Ct formula) [22]. 2.4. Sample Size A sample size of 22 participants allowed ≥80% power to detect significant inter-group differences of 0.26 units in the relative quantification of ATP-binding cassette, subfamily A1 (ABCA1) expression, considering a two-sided type I error of 0.05, a dropout rate of 10%, and the standard deviation of the differences of ABCA1 gene expression after an analogous intervention with a PC-rich VOO [22]. 2.5. Statistical Analysis We evaluated the normality of continuous variables by normal probability plots. We discarded differences in baseline parameters between the eligible volunteers and the whole study population with chi-squared tests, t-tests, and Mann-Whitney U tests (in categorical, normally, and non-normally distributed continuous variables, respectively). We assessed the effects of each intervention relative to baseline by paired t-tests, and the inter-treatment differences in mixed models adjusted for age and sex, in which we also studied period-by-treatment interactions to discard possible carry-over effects. To study whether the differences in gene expression variables after the oil interventions were related among them, we assessed the associations among post-intervention changes by Spearman’s correlation Nutrients 2019, 11, 1732 4 of 10 analyses. We considered significant any p-value < 0.05 and performed all statistical analyses in R Software (Vienna, Austria) v3.1.0 [24]. 3. Results 3.1. Study Participants The 22 eligible volunteers presented no differences in their baseline characteristics relative to the whole trial population (Table 1). Table 1. Study population. Sub-Sample for Whole VOHF Study Transcriptomic p-Value Population (N = 33) Analyses (N = 22) Age (years) 54.8 ± 8.94 55.5 ± 9.98 0.779 Male sex (n, %) 8 (36.4%) 14 (42.4%) 0.866 Glucose (mg/dL) 91.7 ± 13.5 90.8 ± 11.7 0.798 Total cholesterol (mg/dL) 227 ± 31.8 226 ± 35.2 0.968 HDL cholesterol (mg/dL) 31.1 ± 9.21 31.3 ± 10.4 0.952 LDL cholesterol (mg/dL) 171 ± 30.4 170 ± 32.2 0.906 Triglycerides (mg/dL) 116 [91.0; 139] 114 [88.0; 141] 0.790 Systolic blood pressure (mmHg) 128 ± 15.1 128 ± 14.6 0.863 Diastolic blood pressure (mmHg) 75.0 ± 10.8 73.2 ± 10.0 0.536 Body mass index (kg/m2 ) 26.6 ± 4.00 26.6 ± 4.45 0.999 Smokers (n, %) 1 (4.55%) 2 (6.06%) 1.000 Variables are expressed as percentages (categorical variables), means ± standard deviation (normally distributed variables) or median [1st; 3rd quartile] (non-normally distributed variables). 3.2. Gene Expression Analyses FVOOT intake upregulated significantly the expression of cytochrome P450, subunit 27A1 (CYP27A1; p = 0.041), liver X receptor beta (LXRβ; p = 0.005), peroxisome-proliferator activated receptor beta/delta (PPARβ/δ; p = 0.038), and retinoid X receptor alpha (RXRα; p = 0.005), and the expression of ABCA1 in a marginally significant way (p = 0.099), relative to baseline in all cases. Upregulation of caveolin-1 (CAV-1), CYP27A1, and LXRβ was marginally significant relative to the VOO control intervention (p = 0.070, p = 0.053, and p = 0.070, respectively). Finally, VOO intake downregulated RXRβ expression relative to baseline (p = 0.025) (Table 2). CAV1, CYP27A1, LXRβ, PPARβ/δ, and RXRα upregulation after the FVOOT intervention were intimately inter-correlated and associated with increments in ABCA1 expression (r > 0.55, p < 0.0042 -adjusted p-value for 12 comparisons, according to the Bonferroni method-) (Figure 1). Nutrients 2019, 11, 1732 5 of 10 Table 2. Gene expression changes after olive oil interventions. FVOOT Intervention FVOO Intervention VOO Intervention Post- vs. Pre- Post- vs. Pre- Post- vs. Pre- Post-Intervention FVOOT vs. Post-Intervention FVOO vs. Post-Intervention Intervention Intervention Intervention Value VOO p-Value Value VOO p-Value Value Difference Difference Difference ABCA1 0.82 ± 0.60 0.23 ± 0.14 0.402 0.92 ± 0.59 0.15 ± 0.14 0.666 0.87 ± 0.65 0.064 ± 0.14 ABCG1 0.63 ± 0.79 −0.032 ± 0.10 0.588 0.75 ± 0.79 −0.048 ± 0.10 0.663 0.74 ± 0.77 −0.11 ± 0.11 CAV1 1.43 ± 0.93 0.16 ± 0.15 0.070 1.37 ± 1.02 −0.19 ± 0.15 0.814 1.63 ± 1.02 −0.24 ± 0.15 CYP27A1 −0.89 ± 0.81 0.48 ± 0.23 * 0.053 −0.79 ± 0.90 0.24 ± 0.23 0.216 −0.94 ± 0.81 −0.17 ± 0.24 NR1H2 (LXRβ) 0.57 ± 0.6 0.57 ± 0.20 ** 0.070 0.57 ± 0.61 0.052 ± 0.20 0.977 0.62 ± 0.59 0.043 ± 0.21 NR1H3 (LXRα) 0.79 ± 0.51 −0.055 ± 0.12 0.448 0.74 ± 0.59 −0.22 ± 0.12 0.850 0.91 ± 0.58 −0.18 ± 0.12 PPARA 0.14 ± 0.44 0.14 ± 0.12 0.249 0.20 ± 0.52 0.018 ± 0.12 0.661 0.28 ± 0.48 −0.06 ± 0.13 PPARD −0.37 ± 0.56 0.23 ± 0.11 * 0.206 −0.30 ± 0.60 0.12 ± 0.11 0.573 −0.32 ± 0.52 0.029 ± 0.11 PPARG −0.11 ± 1.11 0.19 ± 0.20 0.353 −0.12 ± 1.12 0.20 ± 0.20 0.330 −0.12 ± 1.18 −0.079 ± 0.21 RXRA 0.22 ± 0.54 0.62 ± 0.21 ** 0.132 0.24 ± 0.58 0.071 ± 0.22 0.790 0.32 ± 0.60 0.15 ± 0.22 RXRB 0.76 ± 0.63 −0.050 ± 0.16 0.166 0.80 ± 0.67 −0.16 ± 0.16 0.368 0.89 ± 0.69 −0.37 ± 0.16 * SCARB1 0.22 ± 0.69 0.12 ± 0.10 0.367 0.19 ± 0.76 0.018 ± 0.10 0.845 0.31 ± 0.72 −0.010 ± 0.10 ABCA1: ATP-binding cassette A1; ABCG1: ATP-binding cassette G1; CAV1: caveolin-1; CYP27A1; cytochrome P450, family 27, subunit A1; FVOO: functional virgin olive oil enriched with olive oil phenolic compounds (500 mg/kg); FVOOT: functional virgin olive oil enriched with olive oil (250 mg/kg) + thyme phenolic compounds (250 mg/kg); NR1H2-LXRβ: liver X receptor beta; NR1H3-LXRα: liver X receptor alpha; PPARA: peroxisome proliferator-activated receptor alpha; PPARD: peroxisome proliferator-activated receptor beta/delta; PPARG: peroxisome proliferator-activated receptor gamma; RXRA: retinoic X receptor alpha; RXRB: retinoic X receptor beta; SCARB1: scavenger receptor B-1; VOO: natural virgin olive oil (80 mg/kg). Variables are expressed as means ± standard errors of the mean. Intra-treatment comparisons: *: p < 0.05; **: p < 0.01. Nutrients 2019, 11, 1732 6 of 10 Nutrients 2019, 11, 1732 6 of 10 Figure 1. Significant Spearman’s correlation values of differences after the FVOOT intervention (adjusted for multiple comparisons according to the Bonferroni correction method, p < 0.0042). Figure 1. Significant Spearman’s correlation values of differences after the FVOOT intervention 4. (adjusted for multiple comparisons according to the Bonferroni correction method, P < 0.0042). Discussion In the present work, we report that the consumption of a functional VOO, enriched with olive oil 4. Discussion and thyme PCs, enhanced the expression of key cholesterol efflux genes. In the Thepresent ability ofwork, HDLwe toreport promote thatcholesterol the consumption of a functional efflux from macrophages VOO, enriched is the with olive most studied HDL oil function and thyme andPCs, enhanced reflects the expression of HDL anti-atherogenic key role cholesterol better than HDL efflux genes. levels [25]. Lipid-poor and cholesterol The HDLs small abilitymediate of HDLcholesterol to promoteeffluxcholesterol effluxtofrom by binding ABCA1 macrophages (in a cellularis the systemmostintimately studied HDL related function and reflects HDL anti-atherogenic role better than HDL to caveolae/CAV1 gene expression in immune cells such as macrophages) [26] and large cholesterol levels [25]. Lipid-poor HDLs exert andthis small HDLs process viamediate cholesterol ATP-binding cassetteefflux G1 andby binding scavenger to receptor ABCA1 B1. (in aExpression cellular system of theseintimately cholesterol related to caveolae/CAV1 gene expression in immune cells such as macrophages) transporters is mainly regulated by LXRs and PPARs, key transcription factors in the overall regulation[26] and large HDLs exert this process via ATP-binding cassette G1 and scavenger receptor of lipid metabolism [25,26]. In our study, consumption of the FVOOT functional oil promoted B1. Expression of theseLXR cholesterol transporters gene expression is mainlythrough and activation regulatedtwoby LXRs andpathways hypothetical PPARs, key transcription (Scheme 1): (1) thefactors in the of upregulation overall CYP27A1regulation of lipid metabolism (a monooxygenase member [25,26]. of the In our study,P450 cytochrome consumption of the superfamily), FVOOT enzyme functionalfor responsible oil the promoted LXR gene transformation of expression and oxidized cholesterol into activationsterols, through two hypothetical essential pathways activator ligands (Scheme of LXR; and (2) 1):the (1) upregulation the upregulation of CYP27A1 (a monooxygenase member of the cytochrome of RXRα, whose correspondent protein is necessary to build the LXR-RXR heterodimer P450 superfamily), enzyme responsible (the active complex for the transformation responsible of cholesterol for LXR nuclear functions) into[27]. oxidized sterols, essential A quantitative proteomic activator approach ligands of LXR; performed in and (2) thehad our study upregulation of RXRα, already reported an whose increasecorrespondent in the levels ofprotein severalisproteins necessary to build related to the theLXR/RXR LXR-RXR heterodimer (the active complex responsible for LXR nuclear functions) axis after the functional and the control VOO interventions [28]. Activation of LXR function [27]. A quantitative proteomic approach performed in our study had already reported has been consistently linked to increments in the expression of cholesterol transporters involved in the an increase in the levels of phenomenon efflux several proteins (suchrelated as ABCA1to theand LXR/RXR ABCG1)axis [25] after and ofthe functional CAV1 [26]. Inand the control addition, FVOOT VOO intake interventions [28]. Activation of LXR function has been consistently linked upregulated PPARβ/δ, another transcription factor known to induce the expression of ABCA1 [29] to increments in theand expression CAV1 [30]. of Increases cholesterol transporters in the expression involved of RXR mayin thebeefflux phenomenon additionally related (such as ABCA1 to a greater and of activation ABCG1) [25] and of CAV1 [26]. In addition, FVOOT intake upregulated this PPAR (its active form is also a heterodimer with RXR), which, in turn, is also known to promote PPARβ/δ, another transcription LXR function factor [29].known to induce the expression of ABCA1 [29] and CAV1 [30]. Increases in the expression of RXR may be additionally related to a greater activation of this PPAR (its active form is also a heterodimer with RXR), which, in turn, is also known to promote LXR function [29]. Nutrients 2019, 11, 1732 7 of 10 Nutrients 2019, 11, 1732 7 of 10 Scheme 1. Scheme 1. Modulation Modulationofofcholesterol cholesterolefflux-related transcriptional efflux-related regulators transcriptional due to regulators a functional due virgin to a functional olive oil virgin enriched olive in oliveinoil oil enriched andoil olive thyme phenolic and thyme compounds. phenolic compounds. Olive oil Olive oiland and thyme thyme PCsPCs arethe are not notonlytheknown only to known be abletotobe able tocholesterol modulate modulatemetabolism- cholesterol metabolism-related related gene expression gene expression pathways. pathways. Other dietary Other bioactive bioactivecompounds dietary compounds such as curcumin such as curcumin and 6- and 6-dihydroparadol from ginger, and coenzyme Q10 have dihydroparadol from ginger, and coenzyme Q10 have been shown to promote cholesterol been shown to promote cholesterol efflux- efflux-related related gene expression gene expression in previous in previous in vitro in vitro or orin in vivostudies vivo studies[31,32]. [31,32]. Furthermore, Furthermore, nutritional nutritional supplements of supplements of green green tea catechins have tea catechins also been have also been associated associated withwith aa promotion promotion of of the RXR expression the RXR expression in animal models [33]. Finally, coenzyme Q10 treatment promoted in animal models [33]. Finally, coenzyme Q10 treatment promoted ABCG1-mediated macrophage ABCG1-mediated macrophage cholesterol efflux cholesterol effluxinina arandomized randomized trial in healthy trial in healthyvolunteers volunteers[34].[34]. Upregulation Upregulation of some of these of some of genes these (PPAR-related, ABCA1, and SRB1) genes (PPAR-related, ABCA1, and SRB1) has also been reported after an acute consumption of aVOO has also been reported after an acute consumption of a PC-rich PC- in pre-/hypertensive rich subjects [22]. VOO in pre-/hypertensive However, subjects in the present [22]. However, in thestudy, the study, present FVOOTthe intervention promoted FVOOT intervention the expression promoted of cholesterol-efflux the expression regulatory regulatory of cholesterol-efflux genes only genes versusonlytheirversus baseline, although their baseline, marginally although significant increases in their expression were also observed relative marginally significant increases in their expression were also observed relative to the controlto the control intervention. Differences among intervention. studiesamong Differences can be explained studies can by be theexplained extremely by high content the in PCshigh extremely administered content ininPCs the previous work [22], the study design (acute use versus mid-term sustained administered in the previous work [22], the study design (acute use versus mid-term sustained consumption), and the pathophysiology consumption), of and the participants (hypertensive versus the pathophysiology of the hypercholesterolemic). participants (hypertensive versus The greatest strength hypercholesterolemic). of this study is its randomized, crossover, double-blind, controlled design, which confers strong inter-individual variability control. However, The greatest strength of this study is its randomized, crossover, double-blind, it also presents limitations: controlled the design, observed effects were expectedly modest, since our trial was based on oil doses which confers strong inter-individual variability control. However, it also presents limitations: thethat could be achievable via a regular observed diet,were effects and the control intervention expectedly modest, since wasoura VOOtrial (with a considerable was based amount on oil doses thatofcould olive oil be PCs, 80 ppm), which makes it more difficult to observe inter-group differences. achievable via a regular diet, and the control intervention was a VOO (with a considerable amount of olive oil PCs, 80 ppm), which makes it more difficult to observe inter-group differences. 5. Conclusions 5. Conclusions A functional VOO, enriched with olive oil and thyme PCs (250 mg/kg of each), promoted the expression of several genes regulating HDL cholesterol efflux capacity (CAV1, CYP27A1, LXRβ, PPARβ/δ, A functional VOO, enriched with olive oil and thyme PCs (250 mg/kg of each), promoted the RXRα). Such an improvement may contribute to explaining the beneficial effect of this functional food expression of several genes regulating HDL cholesterol efflux capacity (CAV1, CYP27A1, LXRβ, product on HDL functional capacities in hypercholesterolemic patients. These results contribute to PPARβ/δ, RXRα). Such an improvement may contribute to explaining the beneficial effect of this explaining the beneficial effects of these functional oils towards a more cardioprotective lipid profile. functional food product on HDL functional capacities in hypercholesterolemic patients. These results contribute to explaining the beneficial effects of these functional oils towards a more cardioprotective lipid profile. Nutrients 2019, 11, 1732 8 of 10 Author Contributions: Conceptualization, R.S., M.J.M., M.I.C., M.F. (Montserrat Fitó) and Á.H.; Data curation, S.A., I.S., Á.H. and O.C.; Formal analysis, S.A., I.S., M.F. (Montserrat Fitó), Á.H. and O.C.; Funding acquisition, R.S., M.J.M., M.I.C. and M.F. (Montserrat Fitó); Investigation, M.F. (Marta Farràs), M.F. (Montserrat Fitó), Á.H. and O.C.; Methodology, M.F. (Marta Farràs), S.A., S.C., D.M.-A., G.B., M.K., L.R., S.F.-C. and A.P.; Project administration, R.S., M.J.M., M.I.C. and M.F. (Montserrat Fitó); Resources, M.F. (Montserrat Fitó); Software, S.A., I.S., Á.H. and O.C.; Supervision, M.F. (Montserrat Fitó), Á.H. and O.C.; Validation, S.A., I.S., M.F. (Montserrat Fitó), Á.H. and O.C.; Visualization, M.F. (Marta Farràs) and Á.H.; Writing—original draft, M.F. (Marta Farràs), M.F. (Montserrat Fitó) and Á.H.; Writing—review & editing, M.F. (Marta Farràs), S.A., S.C., I.S., D.M.-A., G.B., M.K., R.S., M.J.M., J.C.E.-G., L.R., S.F.-C., A.P., R.E., M.I.C., M.F. (Montserrat Fitó), Á.H. and O.C., M.F. (Montserrat Fitó), Á.H., and O.C. had primary responsibility for final content. Funding: This research was funded by: the Spanish Ministry of Economy and Competitiveness (AGL2009- 13517-C03-01, AGL2012-40144-C03-01), the Instituto de Salud Carlos III (CB06/03/0028, CB07/08/0016, CD17/00122, CD17/00233, and JR17/00022), FEDER funds (CB06/03), and the Agència de Gestió d’Ajuts Universitaris i de Recerca (2014-SGR-240, 2017-SGR-522). Acknowledgments: We thank Stephanie Lonsdale for her help in editing the English text. CIBEROBN, CIBERDEM, and CIBERESP are initiatives of the Instituto de Salud Carlos III. Conflicts of Interest: The authors declare no conflict of interest. References 1. Martín-Peláez, S.; Covas, M.I.; Fitó, M.; Kušar, A.; Pravst, I. Health effects of olive oil polyphenols: Recent advances and possibilities for the use of health claims. Mol. Nutr. Food Res. 2013, 57, 760–761. [CrossRef] [PubMed] 2. Lou-Bonafonte, J.M.; Arnal, C.; Navarro, M.A.; Osada, J. 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