Association of hydroxytyrosol enriched olive oil with vascular function in chronic coronary disease

Hydroxytyrosol reduces low‐density lipoprotein oxidation, contributing to prevention of atherosclerosis progression.


| INTRODUCTION
Mediterranean diet (MD) is known for its beneficial effects on cardiovascular function. 1 There is evidence that extra-virgin olive oil intake in the context of a MD is associated with a reduction in the risk of cardiovascular disease. 1,2 Extra-virgin olive oil (EVOO) contains large amounts of monounsaturated fatty acids (98%-99% of the total weight of EVOO) and polyphenols, such as hydroxytyrosol, which are associated with its beneficial properties on cardiovascular function. 3 However, it should be noted, that the intestinal absorption rate of olive oil phenols ranges from 40% to 95%. 4 Taking this into account, even with a consumption of 50 g per day olive oil the dietary intake of hydroxytyrosol is estimated to approximately 2 mg. 5 For the above reasons, the intake of oral nutritional supplements containing olive oil enriched with phenolic compounds have become popular, as these provide in small amounts the maximum beneficial antioxidant actions. 6 According to European Food Safety Authority (EFSA), a minimum of 5 mg of hydroxytyrosol in a maximum of 20 g of olive oil should be consumed to protect LDL particles from oxidation. 4 Hydroxytyrosol (HT) and its corresponding derivatives constitute about 90% of the total phenolic content of virgin olive oil. 6 Hydroxytyrosol is an amphipathic phenol with a molecular weight of 154.16 g/mol and a phenylethylalcohol structure. It is present at a very high concentration in the leaves of the olive tree. 7 Experimental and clinical trials have reported the antioxidant, anti-inflammatory and antiatherogenic effects of HT. 7,8 Experimental in vivo studies support that HT enhances protection of low-density lipoprotein (LDL) from oxidation, which might have a possible role in the prevention of atherosclerosis progression. 8 Furthermore, the administration of HT in rats significantly decreased the serum levels of total cholesterol (TC), triglycerides (TG) and LDL, increased the serum level of high-density lipoprotein (HDL), [9][10][11] decreased fasting glucose and improved insulin tolerance. 12,13 Additionally, HT reduced the serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels in mice 13 and had comparable action with aspirin in inhibiting of collagen-induced platelet aggregation in whole blood. 14 Previous studies have shown a beneficial effect of olive oil extract supplements on endothelial function in subjects without known cardiovascular disease. 15 However, the effects of olive oil supplements on cardiovascular function in patients with chronic coronary artery syndrome (CCAS) have not been fully defined.
In the present study, we hypothesized that administration of an oral supplement containing olive oil extract enriched with hydroxytyrosol (OOHT) may improve lipid profile, reduce oxidative burden and improve cardiac and vascular markers in patients with CCAS. Thus, in a prospective, randomized, crossover, double-blind, placebocontrolled, clinical trial, we randomly assigned patients with CCAS to an oral supplement of olive oil extract enriched with HT or placebo for 1 month and we assessed endothelial function and glycocalyx integrity, arterial elastic properties, coronary artery flow reserve and cardiac function as well as oxidative stress, inflammatory biomarkers and lipid profile at baseline and after treatment.
According to the Study design, (Clini calTr ials.gov, NCT04520126) the primary outcome of the study is the effect of OOHT on endothelial glycocalyx thickness, endothelial function, arterial stiffness, coronary function and left ventricular function of patients with CCAS. The study was approved by the Attikon University Hospital scientific ethics committee (Approval number: 32/18-01-2019) funded by OliveHeart Operational Programme Competitiveness, Entrepreneurship and Innovation 2014-2020 (EPAnEK) and by the Hellenic Society of Lipidology of Atherosclerosis and Vascular Disease. The study was conducted according to the Declaration of Helsinki, and written informed consent was provided by the participants. Our study was registered on https://clini caltr ials. gov/, NCT04520126, 17/08/2020.

| Study population
In this study, we recruited men or women between 18 and 70 years old diagnosed with CCAS, as defined from current guidelines. 16 Patients who experienced an acute coronary syndrome and have undergone percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) could be included in the study if 2 or more months have passed since the event.
We excluded from the study elderly patients aged >70 years old and patients diagnosed with diabetes mellitus, uncontrolled hypertension, chronic renal failure (creatinine >2 mg/dL), liver, thyroid and inflammatory diseases affecting inflammatory markers and patients with active malignancies or those treated chronically with corticosteroids. Finally, patients who had joined a weight loss program or underwent any nutritional intervention and strenuous exercise within the previous 2 months and/ or concurrently taking multivitamin preparations with antioxidant properties excluded from recruitment.
Patients were randomly assigned to 2 capsules of an oral supplement containing 2.5 mg HT and 412.5 mg olive oil extracts po twice daily (Olivomed, Intermed) or placebo (OOHT or Placebo) and after 1-month treatment were crossed over to the alternate treatment (Placebo or OOHT) for 1 month (Figure 1). We selected a dose of 10 mg/day for HT, as data from a recent study showed that 15 mg ΗΤ was effective for weight loss and visceral fat mass loss, whereas the dose of 5 mg had no effect, implying the need of increased HT dosage to succeed a sufficient antioxidant and anti-inflammatory effect. 17 The active compound of the oral supplement contained olive oil enriched with 2.5 mg hydroxytyrosol in each capsule. Placebo capsules contained olive oil and had the same label, taste, shape and colour with the OOHT capsules. A dedicated study nurse was responsible for randomization and delivery of boxes of capsules without knowledge of the tests' results.
All researchers and patients were blinded during data acquisition and data analysis.
Assessment was performed at baseline, 1 month after randomization and treatment with OOHT or placebo and 1 month after crossover to placebo or OOHT, respectively ( Figure 1).
Forty-two consecutive patients were screened. However, six patients were excluded because of uncontrolled hypertension (n = 3) or chronic renal failure (creatinine >2 mg/dL, n = 3), and six patients denied participation in the study. All subjects were excluded before assignment to treatment and none after. As a result, 30 patients were finally included in the study ( Figure 1).
We performed blood sampling in the morning of each visit. After blood sampling, we performed initially the vascular studies and then the echocardiography assessment with the CFR evaluation as the final examination. Before the assessment, all subjects abstained from alcohol, caffeine and food for 8 h and ceased taking any vasoactive medications for 24 h. Study treatment was last taken in the evening before the scheduled vascular and cardiac studies. Thus, no acute effect of OOHT on the examined markers would be expected. Patients were instructed to keep their dietary habits stable and to follow a Mediterranean diet scheme 2 throughout the study. Special instructions were F I G U R E 1 Consort flow diagram, study design and study population.
given to consume olive oil as defined by Mediterranean diet 2 and not to exceed consumption of 40 g of olive oil per day. No one of the patients reported a deviation from the dietary instructions given.

| Endothelial function
Brachial artery FMD was ultrasonically assessed in line with published methodology recommendations 18 and expressed as the percent increase of the baseline arterial diameter. Intraobserver variability of the brachial artery diameter was .1 ± .12 mm.

| Endothelial glycocalyx
We measured the perfused boundary region (PBR) of the sublingual arterial microvessels (ranged from 5 to 25 μm) using Sidestream Darkfield Imaging (Microscan, Glycocheck, Microvascular Health Solutions Inc., Salt Lake City, UT, USA). This technique provides a direct, noninvasive and fast method for the assessment of the endothelial glycocalyx. The PBR is the cell-poor layer, which results from the phase separation between the flowing red blood cells (RBC) and plasma on the surface of the microvessel lumen. The PBR includes the most luminal part of glycocalyx that does allow cell penetration. Thus, an increased perfused boundary region is consistent with deeper penetration of erythrocytes into glycocalyx, indicating a loss of glycocalyx barrier properties and is a marker of reduced glycocalyx thickness. The measurement of endothelial glycocalyx thickness using Sidestream Darkfield imaging is easy to perform (duration of 3 min) and is not biased by operator skills. Moreover, it has a standardized methodology, provides measurements of multiple sample sites (>3000 vascular segments of sublingual microvasculature), has very good reproducibility 19,20 and thus is proposed as a valid method to assess endothelial integrity by the European Society of Cardiology Working Group on Peripheral Circulation. The PBR measurements are independent of red blood cell filling (RBC) of the vessels segments (haematocrit), because the software only includes vessel segments that have a filling percentage of more than 50%. Hence, vessel segments are only selected when at least 11 of the 21-line markers have a positive signal for the presence of an erythrocyte. Thus, PBR values are independent of haematocrit, reflecting a damaged glycocalyx that is more accessible for circulating erythrocytes.
Vascular perfused density (mm/mm 2 ) can be determined from the number of vascular segments containing RBCs multiplied by vascular segment length (10 μm). All detected RBC-containing vascular segments with a diameter between 4 and 25 μm were automatically counted in the video recordings of each subject, and total microvascular density was calculated (mm/mm 2 ). Total microvascular density was normalized to tissue surface area.

| Arterial stiffness
Carotid-to-femoral PWV was estimated according to a previous published methodology (Complior, Alam Medical, Vincennes, France), 18 by computing the ratio of the distance between the carotid and femoral pulse palpation site to the pulse wave transit time (m/s). The intraobserver variability was 6%, in accordance with existing evidence in similar studies. 21

| Echocardiography
Studies were performed using a Vivid E95 (GE Medical Systems, Horten, Norway) ultrasound system. All studies were digitally stored in a computerized station (Echopac 204 GE, Horten, Norway) and were analysed by two observers, who had no access to clinical and laboratory data.
LV dimensions and volumes, LV systolic and diastolic properties, LV mass and relative wall thickness (RWT) were estimated according to current recommendations. LA volume was evaluated using the biplane Simpson technique (2-and 4-chamber apical views) and was indexed to body surface area. 22

| Coronary flow reserve
CFR was measured by transthoracic Doppler echocardiography, analysing colour-guided pulse-wave Doppler signals derived from long axis apical projections. The maximal velocity end velocity-time integral in the distal left anterior descending artery were recorded at rest and following intravenous adenosine administration, according to published methodology 23 and quantified as the ratio of hyperaemic to resting maximal diastolic velocity. Coronary flow reserve assessment of left anterior descending artery was feasible in all subjects of the study.

| Doppler echocardiography
The early mitral inflow E wave and late A wave were measured by pulsed-wave Doppler. The deceleration time (DT) of E mitral wave was measured in Doppler mitral inflow recordings. Myocardial velocities were recorded with tissue Doppler imaging. The sample volume was placed in the septal and lateral sites of the mitral annulus in the apical 4-chamber view to record the LV systolic velocity (S′) and early diastolic velocity (E'). The average value of the velocities at the 2 annular sites was used. The ratio of the mitral E the E' (E/E') was also calculated.

| LV myocardial deformation
In all patients, we measured longitudinal systolic strain (LS) from standard 2-dimensional acquisitions (frame rate: 70-80/s) with the use of a dedicated software (EchoPac 204 PC, GE Healthcare). Global longitudinal strain (GLS) was calculated using the 17 LV segment model imaged from apical chamber views (4-, 2-and 3-chamber view), as previously published. 22 The myocardial deformations at the basal, mid-ventricular and apical segments were averaged to GLS. All variables represent the mean value of measurements taken in three consecutive cardiac cycles. Inter-and intraobserver variability of GLS was 8% and 5%, respectively.
Human Oxidized Low-Density Lipoprotein (Human Ox-LDL) was determined spectrophotometrically with a commercial Elisa kit (Elabscience, Houston, Texas, enzyme linked immunosorbent assay kit for determination of Ox-LDL levels; measurement range 62.5-4000 pg/ mL). For Ox-LDL, the intra-assay variability was 5.41%, respectively, and the inter-assay variability was 4.88%, respectively.
Human Apolipoprotein a (Human LP-a) was determined spectrophotometrically with a commercial Elisa kit (Elabscience, Houston, Texas, enzyme linked immunosorbent assay kit for determination of LP-a levels; measurement range .23-15 ng/mL). For LP, the intra-assay variability was 3.34%, respectively, and the inter-assay variability was 4.89%, respectively.
Human C-Reactive Protein (Human CRP) was determined spectrophotometrically with a commercial Elisa kit (Elabscience, Houston, Texas, enzyme linked immunosorbent assay kit for determination of CRP levels; measurement range .39-25 ng/mL). For CRP, the intra-assay variability was 3.57%, respectively, and the inter-assay variability was 3.85%, respectively.

| STATISTICAL ANALYSIS
SPSS v.22 was used to analyse the data. Data are presented as mean ± SD or median with interquartile range (25th-75th percentile), if not normally distributed. The Shapiro-Wilk test was used to examine whether the data were normally distributed, whereas the Levene test was used to examine the homoscedasticity of the data. Nonparametric variables were transformed into rank for analysis. In all analyses, we used two tailed tests with p < .05.
A p value of<0.05 was considered as statistically significant.
Differences between values for each of the measured variables were assessed by independent samples t-test or paired t-test for continuous variables with normal distribution, by Mann-Whitney test or Wilcoxon test for continuous variables with non-parametric distribution and by a chi-squared or Fisher's exact test for categorical variables as appropriate.
Analysis of variance (anova) for repeated measurements was performed for measurements of the examined markers at baseline, 1 month after treatment with placebo and 1 month after treatment with active product used as a with-in-subject factor. The F and p values of the comparison between treatments were also calculated. When the sphericity assumption, as estimated by Mauchly's test, was not met, the Greenhouse-Geisser correction was used. Baseline variables that were of clinical significance, namely age, sex, risk factors, medication and the order of intervention (active compound first followed by placebo versus placebo first followed by active compound) were included in multivariate models as covariates. Paired ttest was performed to compare the percent changes of the examined biomarkers after treatment (OOHT versus placebo).
Based on 1% ± 1% (mean ± SD) and 5% variability 24 for the measurement FMD and coronary flow velocities, respectively, we assumed that a 25% change in vascular markers was clinically significant. In a previous study, 23 the mean absolute change in CFR after intervention was −.86 (range −1.36-−.37; 23% change). Thus, with a mean difference of .86, a variability of the difference of 1.36, an a = .05 (2-sided), and a power of 80%, the sample size needed was calculated to be at least 23 patients for withinpatient comparisons. In previous studies, 19 the mean absolute change in FMD after intervention was 2.5%, and the variability of this change was 3%; thus, with an a = .05 (2sided) and a power of 80%, the required sample size was calculated to be at least 13 patients.

| RESULTS
Thirty patients (27 male, 90%) were finally studied with a mean age 61.6 ± 8.3 years old. All clinical and demographic characteristics of patients investigated are included in Table 1.

| Vascular markers
At baseline, decreasing values of FMD were related to increasing values of PWV (r = −.422, p = .030) and decreasing values of CFR (r = .525, p = .028).
Compared to baseline, treatment with OOHT improved PWV (11.1 ± 1.8 vs. 11.8 ± 2.3 m/s, p = .002), while no significant effect was observed after treatment with placebo (11.4 ± 2.2 m/s, p = .290). Moreover, the percent reduction in PWV after treatment with OOHT was greater than the respective PWV reduction after placebo (5.3% vs. .3%, p = .006). Augmentation index, central systolic BP, central diastolic BP and central pulse pressure remained similar throughout the study (p > .05 for all comparisons).

| Echocardiographic markers
Treatment with OOHT but not with placebo improved LV diastolic function markers 1-month post-treatment, compared to baseline. Deceleration Time E' wave ( Figure 2)
T A B L E 2 Comparison of Vascular markers at baseline, after 1-month treatment with HT, after 1-month treatment with placebo. By anova, there was no significant interaction between sex or order of intervention and changes of the measured markers post-treatment (p > .05).

| DISCUSSION
In the current, prospective, double-blind, crossover and placebo-controlled clinical trial, we have shown that administration of an oral supplement containing an olive extract enriched with hydroxytyrosol (10 mg hydroxytyrosol po daily) resulted in a significant improvement in vascular function, LV diastolic function and reduced biomarkers of inflammation, oxidative stress and triglyceride levels compared to baseline, while these changes were not evident after treatment with placebo in patients with chronic coronary artery disease.
Two major findings were the reduction in MDA, a marker of lipid peroxidation and CRP, a validated inflammatory marker, in patients treated with OOHT compared to placebo. The changes on inflammatory and oxidative stress burden may explain the parallel improvement of endothelial function, as assessed by FMD and endothelial glycocalyx and all together may have contributed to the observed improved total microvascular density, coronary flow reserve, arterial elasticity and LV after OOHT administration compared to placebo (Figure 3). However, this hypothesis needs to be further validated in future studies.

| Endothelial function
In the present study, we have shown that OOHT administration had a positive impact on endothelial function compared to baseline, while these changes were not observed after treatment with placebo. This stems from the increase in, FMD, CFR and total microvascular density and decrease in PBR (indicating improved glycocalyx integrity) in CAD patients treated with OOHT for 1 month compared to baseline. In the same group of patients, we did not notice a significant shift in the above vascular markers after 1-month treatment with placebo compared to baseline.
In recent studies, it was shown that administration of a supplement containing HT improved markers of atherosclerosis and endothelial function, such as FMD in subjects with mild-to-moderate hypercholesterolaemia. 25 According to another contemporary trial, which studied whether EVOO or butter could influence endothelial function in subjects with type 1 diabetes when added to a single high glycaemic index meal, it was noticed that FMD was significantly improved after EVOO-enriched meal compared with butter. 26 In our study, the reduction in TG after OOHT may have also contributed to improvement of endothelial function. 27 It is well documented that FMD is NO dependent and that NO availability is involved in oxidative status. 20 In our study, we observed improvement of FMD after OOHT implying increased NO availability. Studies support that impaired NO-dependent endothelial function is related to arterial stiffening as assessed by PWV. 28 Thus, we may hypothesize that the reduction in oxidative and inflammatory burden by OOHT administration in the present study resulted to improved endothelial function leading to improved arterial wall properties as assessed by reduction in PWV post-treatment.
Clinical studies also support that OOHT administration reduced oxidative stress leading to improved T A B L E 4 Comparison of biochemical parameters and of lipid profile at baseline, after 1-month treatment with HT, after 1-month treatment with placebo. endothelial function in type II diabetic patients 29 though blood pressure remained unchanged. This is in line with our findings of no profound effect of OOHT on central or brachial blood pressure. Similarly, improvement of oxidative stress and endothelial function leading to improved arterial wall properties may also explain the improved CFR after OOHT treatment. In support of this hypothesis, studies have shown a close link of increased PWV with reduced CFR values in hypertensive and patients with CAD and that reduction in PWV after treating is linked with improve CFR values. 29,30 Indeed, polyphenols contained in red wine have been considered to be responsible for the increase in coronary flow-velocity reserve. 31,32 However, for the first time, we report that administration of OOHT for 1 month may have a favourable impact on coronary microcirculatory function.

| Heart function
In the present study, we have shown that treatment with an oral supplement containing olive oil enriched with HT improved Doppler markers of LV diastolic function, namely E'; E/E and deceleration time in patients with stable CAD. This may be explained by the combined effect of improved arterial elasticity and coronary microcirculatory function on LV function, as observed after treatment with OOHT compared to baseline but not after placebo. Studies support the close link between PWV and CFR with LV diastolic function and the association between improvement of these vascular markers after treatment with the concomitant improvement of LV function. 31 In experimental studies, administration of HT improved the myocardial injury, restored the hemodynamic function and inhibited the angiotensin-converting enzyme (ACE) activity in mice suffered from isoproterenolinduced myocardial infarction. 33 Similarly, oleuropein, one of the main polyphenolic constituents of olive oil, when administered during postconditioning interventions provided robust and synergistic cardio protection in experimental models of ischemia reperfusion injury. 34 Moreover, OOHT hindered deterioration of heart function by minimizing mitochondrial impairment and by reducing oxidative stress after treatment with doxorubicin in rats. 35 Experimental studies have shown that oleuropein administration reduced doxorubicin-induced cardiotoxicity through suppression of oxidative and nitrosative stress. 36 Finally, treatment with HT improved impaired glucose tolerance, endothelial function and decreased left ventricular fibrosis and resultant diastolic stiffness in a diet-induced rat model of metabolic syndrome. 12 Thus, it is possible that reduction in oxidative stress by HT treatment had a direct effect on myocardial function resulting in improved LV diastolic function in our study.
To the best of our knowledge, this is the first study showing benefits of OOHT on cardiac function in patients with CAD.

F I G U R E 3
Major molecular mechanism of the effect of hydroxytyrosol on the coronary artery.

| Underlying mechanisms of antiinflammatory and antioxidant OOHT properties
The anti-inflammatory effects of HT have been widely studied in vitro, in vivo and in clinical studies.
Recently, a sub study of PREDIMED trial, in high-risk subjects for cardiovascular events, reported an inverse relation between urinary polyphenol excretion and plasma concentration of inflammatory biomarkers, namely IL-1β, tumour necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP-1) 37 suggesting a dosedependent anti-inflammatory effect of polyphenols. Moreover, in the VOLOS (Virgin Olive Oil Study) study, subjects with untreated mild dyslipidaemia showed a 20% reduction in serum thromboxane B2 (TXB2) when treated with a phenol-rich olive oil (40 mL/day with high concentration of HT, 166 mg/L) compared to those treated with phenol poor oil (low concentration of HT, 2 mg/L) for 7 weeks. 38 In a study of patients with CCAS, the investigators reported a reduction in proinflammatory cytokines (IL-6) and inflammation markers (CRP) only in those patients randomized to receive 50 mL/day of virgin olive oil rich in HT and not to those administered with 50 mL/day of oil with low concentration of HT for 3 weeks. 39 Mediterranean diet rich in EVOO resulted into reduction in inflammatory biomarkers including IL-6 and CRP. 39,40 Clinical trials that evaluated the effect of different concentrations of olive oil phenolic compounds on inflammatory markers in patients with cardiovascular risk and CAD have provided controversial findings. 41 In our study, we found significant reduction in CRP levels and a borderline reduction in IL-6 levels suggesting an anti-inflammatory effect after short-term treatment with OHHT in patients with CCAS.
PCSK9 plays a critical role in regulating circulating cholesterol levels through the reduction in the membraneassociated LDL receptor. Studies have shown that PCSK9 increases systemic inflammation 42 and has been associated with new onset respiratory and cardiovascular failure at a level >270 ng/mL after infection and is related to extent of CAD. 43 Interestingly in our study, CAD patients had a mean value of 289 ng/mL and elevated PCSK9 blood levels have been also reported in patients with acute myocardial infarction. 44 PCSK9 regulates a wide variety of physiopathological processes, which include lipid metabolism, inflammatory and stress responses, glucose metabolism and cell apoptosis. 45 Regarding the role of PCSK9 in inflammatory process, it is known that PCSK9 causes the stimulation of a set of chemokines and cytokines, specifically from macrophages. This stimulation results in an increased infiltration and activation of monocytes. In addition, vascular smooth muscle cells (VSMCs) are activated by PCSK9, stimulating the conversion to activated macrophages. Interestingly, it has recently been shown that evolocumab (a PCSK9 inhibitor) significantly prevents cytotoxicity induced by hydrogen peroxide and reduces hydroperoxides and MDA levels in human umbilical vein endothelial cells. 46 In line with findings of the above investigations, we have found in our study a reduction in CRP and PCSK9 levels as well as a non-statistically significant trend for reduced IL-6 levels after treatment with OOHT compared to baseline, while placebo did not show similar findings in patients with CCAS treated with statins. These findings suggest that administration of olive extract enriched with HT may have a significant impact on the inflammatory burden of CAD patients and complement the antiinflammatory effect of statins.
Importantly, as also statins may contribute to elevation of PCSK9 levels, 47 the addition of OOHT to the treatment regimen of CAD patients may outweigh any potential adverse effects of statins related to increase of PCSK9 levels.
Studies have shown a positive association between plasma PCSK9 and plasma TG levels. 48 Thus, the reduction in PCSK9 after OOHT administration may have also contributed to the reduction in TG in addition to the direct effect of hydroxytyrosol on lipids.
The antioxidant properties of HT are due to scavenging of radicals and stimulation of synthesis of antioxidant enzymes (such as superoxide dismutase, catalase), which also limit the lipid peroxidation of LDL cholesterol. In the current clinical study, we also observed a significant reduction in MDA, an oxidative stress biomarker reflecting lipid peroxidation and circulating levels of oxidized LDL after OOHT administration.
Additionally, oleuropein, an olive constituent, exhibited significant anti-ischemic and antioxidant properties, as well as reduced the circulating lipids in animal models of experimental myocardial infarction. 49 MDA produced during fatty acid oxidation was used as an index of serum lipid peroxide concentration, while serum 3-Nitrotyrosin concentrations were determined as an index of peroxynitrite. This could be explained by the fact that HT is less potent in scavenging peroxynitrite in comparison with hydroxyl radicals (•OH). 50 Consequently, the short-term intervention (1-month therapy) maybe is not sufficient to obtain the maximum inhibitory effect of HT.
In an experimental model of type 1 diabetic rats, researchers studied the effect of polyphenols in oxidative and nitrosative stress and in platelet activation. HT exerted an antioxidant and downregulatory effect on prothrombotic biomarkers while reducing the fall of prostacyclin. The combination of HT plus dihydroxyphenylglycol (DHPG) showed a more intense effect than each polyphenol alone in the reduction in oxidative and nitrosative stress, platelet aggregation, production of prostacyclin, myeloperoxidase and vascular cell adhesion molecule 1 (VCAM-1). 51 Thus, it may be necessary to combine administration of polyphenols to succeed a more profound effect on nitrosative stress in the clinical setting. This hypothesis may explain the lack of a significant effect of OOHT administration on nitrotyrosin, a marker of nitrosative stress, in our study.
To sum up, most of the studies used experimental models or included patients with risk factors for cardiovascular disease to investigate the antioxidative and inflammatory effects of HT. To our knowledge, in the present study, we provide for the first-time evidence supporting the efficacy of OOHT administration at dose of 10 mg per day to reduce the inflammatory and oxidative stress burden in patients with chronic coronary syndromes.

| LIMITATIONS
Though this study is a prospective, randomized, crossover, double-blind, placebo-controlled, clinical trial, it is a single-centre study that recruited a modest number of subjects and investigated the effects of an olive oil supplement enriched with HT within a short period of time. Sex imbalance might have affected the results of FMD posttreatment, as women have lower FMD values than men 52 ; however, we have adjusted our analysis for sex. The potential causal association of oxidative stress and inflammation with endothelial and vascular function leading to LV diastolic function appears to be a valid hypothesis, though it has not been proven by this study. In the current study, it is not clear whether the observed changes on the vascular and cardiac markers after treatment are caused by HT alone, as we examined only the combination of olive oil with HT and not each compound separately.

| CONCLUSION
Administration of an olive oil supplement enriched with HT improves endothelial and arterial function, reduces oxidative and inflammatory stress after a short period of treatment and improves LV diastolic function in patients with chronic coronary artery syndromes. Whether these beneficial effects have an impact on major cardiovascular events remain to be investigated by future studies.