Resveratrol content in red wine has been recurrently used to justify red wine benefits, such as the so-called ‘French Paradox’, i.e. low mortality due to CVD in France compared to that in other developed countries in spite of sharing CVD risk factors such as obesity, fat intake, smoking, etc. [17]. However, the presence of resveratrol in the diet is almost negligible [18] because it is a minor polyphenol whose content in red wine is low and highly variable [19]. Resveratrol is a defensive compound (phytoalexin) that is synthesized by grapes to face adverse environmental conditions [14] and, by definition, its presence in grapes (and red wine) is low and unpredictable. The vast majority of studies carried out with resveratrol have used the synthetic molecule or non-dietary herbal preparations. Therefore, i) the consumption of wine does not ensure the intake of enough resveratrol to exert beneficial effects, ii) the conjecture that directly correlates the cardiovascular protection of red wine and its resveratrol content is not fully true and iii) the specific contribution of the minor resveratrol content against the rest of major phenolic compounds in red wine is not known. In this regard, we used a resveratrol-rich grape extract (GE-RES) obtained from ultraviolet-treated grapes that induced resveratrol to face such challenge. Therefore, this nutraceutical offers the possibility of including resveratrol in the human diet within its natural edible matrix, the grape. In addition, in order to evaluate the relevance of RES against the rest of the grape phenolics we included a conventional grape extract (GE) group (same grapes without UV treatment).

Adiponectin is an anti-inflammatory cytokine involved in the pathogenesis of vulnerability of coronary lesions by exerting protective pleiotropic effects on the vascular system [20]. Adiponectin release has been reported to be suppressed in the epicardial adipose tissue of patients with obesity and coronary artery disease (CAD) [21]. Despite its anti-inflammatory and protective role against cardiovascular disease (CVD), high adiponectin levels have been correlated with mortality in specific clinical conditions of CAD patients [22]. However, in this case, it has been suggested that higher adiponectin levels are a consequence rather than a cause, i.e. the attempt to protect, reduce or limit endothelial damage provoked by an inflammatory milieu in these patients [23, 24]. In fact, serum adiponectin values have been reported to be lower in CAD patients with vulnerable plaques [25], acute myocardial infarct (AMI) and unstable angina than in those patients with stable angina [26]. In this regard, methods for increasing adiponectin have been suggested as a promising therapy for the prevention and treatment of CAD [27]. Unlike adiponectin, PAI-1 is higher in CAD patients [28] and its reduction can decrease CVD risk because the impairment of fibrinolysis, due to high circulating PAI-1 levels, is associated with the development of AMI [29]. The inverse relation between adiponectin and PAI-1 has also been described in obese [30] and stable angina patients [31]. However, the evolution of these markers is not routinely assayed in the follow-up of CAD patients with optimized medication [32, 33]. In fact, in the present trial, even though patients were being treated according to current evidence-based standards (Table 1), both adiponectin and PAI-1 presented an unfavorable evolution along the trial in the placebo group. Remarkably, the outcomes of our study suggest that a daily, yearlong, dietary intervention with a resveratrol-rich grape nutraceutical increases adiponectin and decreases PAI-1 circulating levels in CAD patients, which is in accordance with the effects exerted by resveratrol in a number of in vitro and animal studies [34]. Other mechanisms related to the cardiovascular protection exerted by resveratrol include the decrease in endothelin-1, adhesion molecules to endothelium, platelet aggregation, and pro-inflammatory cytokines, the increase of NO synthesis and the activation of sirtuins, among others [35].

The specific mechanisms of action reported for GE-RES have been linked to the regulation of genes involved in lipid metabolism and metabolic disorders [36] and a reduction of vascular oxidative stress resulting in the prevention of early atherosclerotic lesions in the aorta of pigs fed with an atherogenic diet [15]. However, despite the abundant preclinical cardiovascular benefits described for resveratrol [35], human clinical trials, especially long-term interventions, are very scarce [11]. Resveratrol consumption has been reported to improve glycemic control in diabetics [37, 38] and endothelial function in CAD patients [39]. However, these studies involved a short follow-up (maximum 3 months) and the statistical analyses did not take into account possible covariates (medication, age, gender, etc.) that could influence the results. Recently, we have shown that a 6-months consumption of GE-RES decreased the concentration of oxidized low-density lipoprotein particles and apolipoprotein B in statin-treated subjects at high CVD risk (primary prevention of CVD) [13]. In the same cohort of subjects, we further reported the improvement of their inflammatory (mainly by decreasing hsCRP) and fibrinolytic (PAI-1) status after one year follow-up [14]. In the present trial, CAD patients were treated with higher statin doses than high-risk subjects in primary prevention (i.e. 20 mg rosuvastatin, 40–80 mg atorvastatin, 40 mg pravastatin or 80 mg fluvastatin). It is known that treatments with high statin doses are correlated with a reduction of CRP [40]. For example, 40 mg pravastatin decreased hsCRP by 13 % (median from 2.7 to 2.4 mg/L) [41] and 18 % (mean from 3.6 to 3.1 mg/L) [42] after 6 months and 5 years, respectively, in secondary prevention cohorts. Therefore, the high statin dose could be behind the low added-effect on hsCRP after consuming GE-RES in this study in comparison with our previous trial in primary prevention of CVD [14]. Nevertheless, the evolution of hsCRP levels in the present trial was clearly better in the GE-RES group than in the other groups (Fig. 2). In contrast to the moderate effect on hsCRP, the effects on PAI-1 and adiponectin upon consumption of GE-RES were clearer in CAD patients than in primary prevention of CVD [14].

Many of the reported effects for adiponectin are related to the inhibition of pro-inflammatory signaling in PBMCs, monocyte adhesion to vascular endothelium and migration into tunica intima, macrophage activation and transformation to foam cell, smooth muscle cell proliferation and migration into the intima [25, 26]. Therefore, we next investigated, through a microarray analysis, whether this dietary intervention could affect inflammatory pathways in PBMCs. Gene expression response to inflammatory signals is mediated through the activation of transcription factors like NF-κB, which contributes to the pathophysiology of inflammatory disease and triggers monocyte recruitment into subendothelial space, a crucial milestone in the pathogenesis of atherosclerosis [43]. Another important transcription factor is AP-1, whose dimers such as JUN/ATF2 are involved in the regulation of diverse subsets of target genes whose products regulate the activation, proliferation, differentiation and apoptosis of leukocytes [44]. In addition, the activity of both NF-κB and AP-1 is regulated by CREBBP [45]. All these transcription factors were predicted to be inhibited following the yearlong consumption of resveratrol-rich grape extract (GE-RES). Furthermore, KLF2, which downregulates PAI-1 expression and negatively regulates pro-inflammatory activation of monocytes through inhibition of NF-κB and AP-1 [4], was predicted to be activated exclusively in the GE-RES group. After combining all the above transcription factors in a joint pathway, a subset of 27 significantly downregulated genes, whose products act in the extracellular space, stood out. The functional analysis of this orchestrated downregulation pointed towards the inhibition of clinically relevant key features in atherothrombosis such as inflammation, cell migration and T-cell interaction [5]. In this regard, Karastergiou et al. [17] reported that the adhesion of monocytes to human endothelial cells was increased by the release of epicardial adipose tissue cytokines in CAD patients. Interestingly, adiponectin reversed these atherogenic effects and this supports the link found in our trial between adiponectin increase and the inhibition of atherothrombotic signals in PBMCs from CAD patients belonging to the GE-RES group.

We next tried to correlate the effects of GE-RES with circulating resveratrol and/or other phenolic metabolites derived from the grape extract GE-RES. Plasma samples were routinely analyzed using state-of-the-art analytical techniques (UPLC-QqQ). However, we were not able to detect circulating resveratrol or other grape-derived metabolites in the GE-RES group. This was in accordance with our previous studies in both humans [13, 15] and pigs [16] using the same grape nutraceutical. At first, this could be explained by the low resveratrol dose assayed (8 mg for 6 months and 16 mg for the last 6 months), by the fast clearance of resveratrol and also because blood withdrawals were carried out after fasting overnight. In this regard, Azorín-Ortuño et al. [46] coined the expression ‘Resveratrol Paradox’ to illustrate the high activity exerted by resveratrol despite its low bioavailability. Circulating resveratrol metabolites were not found in pigs fed with this nutraceutical as stated above. However, a number of resveratrol metabolites were detected in the aortic tissue of these pigs [16]. Although the direct association between resveratrol and/or specific circulating resveratrol metabolites and the biological activity of resveratrol is far from being demonstrated in vivo, this suggests that repetitive, chronic exposure of low resveratrol doses such as those assayed in the present trial could be directly related to the beneficial effects observed. Overall, this implies that each specific product containing resveratrol should demonstrate both its safety and efficacy.