Background

The Mediterranean Diet, as originally defined by Ancel Keys, is characterized by the dietary habits observed in Mediterranean natives during the 1960s, emphasizing low saturated fat and high vegetable oil intake [1,2]. Over time, our understanding of nutrition has evolved, prompting a shift towards investigating total dietary patterns. The concept of a "dietary scheme" has emerged, involving a comprehensive analysis of food and nutrient consumption within specific dietary patterns [3].

Recent research has underscored the pivotal role of the Mediterranean-style diet in preventing cardiovascular diseases. The Lyon Diet Heart Study demonstrated its positive impact on secondary prevention [4]. The landmark PREDIMED study, a large-scale randomized trial, provided compelling evidence for the effectiveness of the Mediterranean-style diet in primary prevention, significantly reducing cardiovascular events compared to a low-fat diet [5–7].

Cardiovascular diseases, including coronary artery disease, cerebrovascular disease, peripheral artery disease, and heart failure, remain the leading global cause of mortality. Atherosclerosis, the main culprit behind these diseases, has a complex pathogenesis involving genetic, metabolic, environmental, and behavioral factors [8]. Hypercholesterolemia, a major metabolic risk factor, contributes significantly to atherosclerosis. Studies consistently highlight the role of reducing fat in the diet, particularly saturated fat, in lowering plasma cholesterol levels and reducing the incidence of coronary heart disease. Notably, the substitution of saturated fat with polyunsaturated and monounsaturated fats, such as those found in virgin olive oil, has shown positive effects [9,10].

Despite established clinical and biochemical risk predictors, a substantial residual risk persists, necessitating the identification of additional predictive biomarkers. Ceramides, belonging to the sphingolipid family, have been implicated in cellular dysfunction and insulin resistance. Excessive de novo ceramide biosynthesis, triggered by stimuli like high serum levels of saturated free fatty acids, may contribute to the pathogenesis of cardiometabolic diseases, including insulin resistance and low-grade inflammation [11,12]. Therefore, evaluating serum ceramide levels becomes crucial in understanding the intricate interplay between lipidaemic pathways and vascular health.

Adipocytes, the cells responsible for storing and releasing fat, produce hormones that play a crucial role in the local microenvironment and distant tissues. Adipokines, specific hormones secreted by adipose tissue, have been linked to inflammation, atherosclerosis, and related cardiovascular complications [13].

Despite advancements in understanding, few studies have prospectively analyzed the association between ceramides and the incidence of cardiovascular and cerebrovascular events. Laaksonen et al. reported an unclear relationship between plasma ceramides and death from cardiovascular disease [14]. Additionally, the PREDIMED study suggested that dietary interventions following a Mediterranean-style diet positively modified the association between a blood ceramide score (comprising individual ceramides and ceramide ratios) and the risk of incident CVD [15]. The CORDIOPREV study in patients with established coronary heart disease concluded that the Mediterranean diet was superior to the low-fat diet in preventing major cardiovascular events in secondary prevention [16].

Despite these findings, no study has comprehensively evaluated the effects of the Mediterranean Diet on endothelial function, in parallel with its impact on serum lipid changes, including ceramide changes, and on specific adipokine pathways. In response to this gap, we have designed a randomized trial to systematically investigate the effects of the Mediterranean diet on endothelial function, measured by the evaluation of the reactive hyperaemia index (RHI), as well as its impact on serum ceramide and adipokine levels. This research aims to provide a holistic understanding of the intricate connections between diet, vascular health, and cardiometabolic risk factors.

Materials and methods

Definition of risk factors

We collected a careful medical history for each patient at the time of hospitalisation, and performed a detailed physical examination. Each subject received routine laboratory tests and M-mode and 2D echocardiogram.

Diabetes was defined according to the ADA 2021 criteria or by a previous dietary, oral antidiabetic or insulin treatment before hospital admission [17].

Arterial hypertension was assessed using the ESC/ESH 2018 criteria [18].

Hypercholesterolemia was defined as serum total cholesterol levels ≥ 5.1 mmol/L, while hypertriglyceridemia was defined as values ≥ 1.7 mmol/L following NCEP-ATP III criteria [19].

The study protocol was approved by the institutional review board, and the study was performed in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice [20].

Each patient provided written informed consent before participation.

The trial has been registered in Clinical Trials.gov [ClinicalTrials.gov Identifier: NCT04873167] on 05/05/2021 and it has been designed and conducted according criteria pre-established by CONSORT (Consolidated Standards of Reporting Trials) (see Fig 1).

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Fig 1. CONSORT 2010 flow diagram.

https://doi.org/10.1371/journal.pone.0300844.g001

Ethics approval and consent to participate.

This protocol study was approved by the Ethics Committee of the Policlinico P. Giaccone Hospital, and all patients provided written informed consent to participate in the study and for sampling and banking of the biological material. Written informed consent was obtained from all the patients. A statement of ethics approval was obtained using the name of the ethics committee and reference number if appropriate

Consent for publication.

All enrolled patients (or legal parents or guardians for children) provided consent to publish individual patient data.

Results

We enrolled 101 patients randomised to the Mediterranean Diet and 52 control subjects randomised to a low-fat diet with a dietary "counselling" between September 2017 and December 2020. No patient was lost at follow-up in both the groups.

Clinical, demographic, and laboratory variables of patients at the time of enrolment are shown in Table 1.

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Table 1. Clinical and laboratory variables at baseline in subjects treated with Mediterranean diet and in control group.

https://doi.org/10.1371/journal.pone.0300844.t001

The univariate analysis showed that subjects in the Mediterranean Diet group showed significantly lower mean creatinine values (1.1 ± 0.4 vs. 1.4 ± 1.2, p = 0.010), significantly higher mean CRP values (11.1 ± 10.8 vs. 5.6 ± 7.3, p = 0.002) and significantly higher ejection fraction (EF) mean values (48.8 ± 8.8 vs. 27.1 ± 26.3, p <0.0005) than those in the control group. With regard to biochemical variables, mean serum levels of resistin was higher among subjects in the Mediterranean Diet group than those in the control group (10.8 ± 1.5 vs. 10.2 ± 1.6, p = 0.037), as well as the values of Cer -24 (2.23 ± 0.19 vs. 2.15 ± 0.21, p = 0.030), Cer-16 (0.15 ± 0.01 vs. 0.14 ± 0.01, p <0.0005) and Cer-22 (0.60 ± 0.03 vs. 0.55 ± 0.04, p <0.0005), with statistically significant inter-group differences. There were also more smokers and patients with chronic kidney disease and carotid plaque among the subjects in the Mediterranean Diet group and the difference was statistically significant.

Table 2 shows the quantitative and qualitative variables of the two groups at baseline, at the six-month (T1) and at the twelve-month (T2) follow-up.

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Table 2. Metabolic and lipidic variables and vascular health indexes in Mediterranean Diet group and control group at baseline (T0), at 6 (T1) and 12 month (T2) follow-ups.

https://doi.org/10.1371/journal.pone.0300844.t002

At the six-month follow-up (T1), subjects in the Mediterranean Diet group showed lower mean serum total cholesterol levels (144 ± 31.9 mg/dl vs. 163 ± 45.9 mg/dl, p = 0.003), compared to the control group (see Table 2).

At the six-month follow-up in subjects in the Mediterranean Diet group, compared to the control group we observed a higher increase in RHI values (2.1 ± 0.4 vs. 1.7 ± 0.6, p <0.0005) (see Table 2).

With regard to biochemical variables, patients in the Mediterranean Diet group also showed lower serum levels of resistin at the six-month follow-up compared to the control group (9.7 ± 1.0 vs. 10.2 ± 1.1, p <0.0005), higher values of adiponectin (28.9 ± 2.9 μg/mL vs. 23.2 ± 4.3 μg/mL, p <0.0005), lower values of visfatin (12.3 ± 1.8 ng/ml vs. 15.1 ± 3.3 ng/ml; p<0.0005), lower values of Cer- 24 (1.96 ± 0.28 μg/mL vs. 2.19 ± 0.23 μg/mL, p <0.0005), higher values of Cer-22 (0.60 ± 0.39 μg/mL vs. 0.57 ± 0.45 μg/mL, p <0.0005) and higher values of Cer-24/Cer-16 ratio (12.11± 0.90 ug/mL vs 11.60 ± 1.13 ug/mL; p = 0.003), with statistically significant inter-group differences (see Table 2). With regard of body weight the mean body weight at six-month follow up were in Mediterranean group vs control group was similar (73.4± 1.1 vs 73.1±0.9).

At the twelve-month follow-up (T2), subjects in the Mediterranean Diet group showed significantly lower serum total cholesterol levels (134 ± 26.7 mg/dl vs. 156 ± 42.8 mg/dl, p<0.0005), lower serum LDL cholesterol (63.4 ± 23.5 mg/dl vs. 74 ± 18.0 mg/dl; p = 0.009), compared to the control group (see Table 2).

At the twelve-month follow-up, we observed a further significant increase in the mean RHI in the subjects of the Mediterranean Diet group (2.2 ± 0.3 vs. 1.7 ± 0.5; p<0.0005), whereas we observed no significant differences in the mean serum levels of adiponectin (23.4 ± 0.4 μg/mL vs. 23. 2 ± 0.4 μg/mL; p = 0.790), a more significant reduction of serum levels of resistin (8.2± 0.9 ng/ml vs. 10.2 ±1.1 ng/ml, p<0.0005) and visfatin (10.2 ± 1.4 ng/ml vs. 14.8 ± 3.8 ng/ml, p<0.0005); no significant change was observed with regard to AIX (see Table 2). With regard of body weight the mean body weight at six-month follow up in Mediterranean group vs control group was similar (73.2± 1.0 vs 73.2±0.9).

At the twelve-month follow-up, the same group also showed significantly lower values of Cer-24 (1.7 ± 0.33 μg/mL vs. 2.7 ± 2.41 μg/mL; p <0.0005), no significant difference in Cer-16 and Cer-22, significant lower serum levels of Cer-18 (0.32 ± 0.01 μg/mL vs. 0.59 ± 0.04 μg/mL p <0.0005) and higher values of Cer-24/Cer-16 ratio (13.34±1.11 ug/mL vs 11.62 ± 0.76 ug/mL; <0.0005) (see Table 2).

In subjects randomized to Mediterranean diet at intragroup analysis we observed a significant change of total cholesterol at T1 and at T2 vs T0 (p<0.0005) with a progressive lowering of serum total cholesterol across the follow-up visits. We also observed a significant intragroup change of LDL cholesterol mean serum levels at T2 vs T0 (p<0.005) with a further reduction of LDL serum level at 12 month follow-up (T2).

In Mediterranean group at intragroup analysis we also observed a progressive lowering of triglyceride serum levels at T1 vs T0 (p<0.05) and a progressive lowering of mean resistin serum levels and of visfatin mean serum levels at T1 and T2 vs T0 (p<0.0005 in both cases), whereas with regard of adiponectin mean serum levels we observed a significant increase of its mean serum levels at T1 vs T 0 (p<0.0005). With regard of ceramides, at intragroup analysis we observed a significant progressive reduction of mean serum levels of C24:0, C22:0, at T2 vs T0 (p<0.0005), and of mean serum levels of C18:0 at T2 vs T0 (p<0.0005), whereas with regard of C24:0/C16:0 we observed a significant increase of its mean serum levels at T1 and T2vs T 0 (p<0.0005).

In subjects randomized to low-fat diet at intragroup analysis we observed a significant change of total cholesterol at T1 and at T2 vs T0 (p<0.0005) with a progressive lowering of serum total cholesterol across the follow-up visits. In low-fat diet group at intragroup analysis we also observed a progressive lowering of triglyceride serum levels at T1and T2vs T0 (p<0.0005), whereas with regard of adiponectin we observed a significant increase of its mean serum levels at T1 and T2 vs T0 (p<0.005). With regard of ceramides, at intragroup analysis we observed a significant increase of mean serum levels of C:22 at T1 and T2 vs T0 (p<0.0005).

At multivariable regression analysis we observed a significant correlation between some clinical and lipidaemic variables and some adipokine and vascular health markers and a beneficial clinical effect of Mediterranean Diet on follow-up. In particular we observed a significant positive correlation between Mediterranean diet with RHI (Exp(B): 5,990; P = 0.0001), and Adiponectin (Exp(B): 1.472; p = 0.0001), and a significant negative correlation with AIX (Exp(B): 0.979; p = 0.032), Total cholesterol (Exp(B): 0.984; p = 0.002) visfatin (Exp(B): 0.675; p = 0.002), and resistin (Exp(B): 0.466; p = 0.005) at six month follow-up (see Table 3). On this light, we also observed a significant positive correlation between Mediterranean diet with RHI (Exp(B): 14,420; P = 0.0001), and a significant negative correlation with AIX (Exp(B): 0.973; p = 0.034), Total cholesterol (Exp(B): 0.982; p = 0.001), LDL cholesterol (Exp(B): 0.983; p = 0.037) visfatin (Exp(B): 0.409; p<0.0005) and resistin (Exp(B): 0.091; p = 0.0001) at 12 month follow-up (see Table 3).

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Table 3. Multivariable regression analysis of the correlations between some clinical variables, vascular health indices, lipidaemic profile and adipokines, and Mediterranean diet effect (independent variable) at the different follow up (0, 6 and 12 months).

https://doi.org/10.1371/journal.pone.0300844.t003

Discussion

Our study revealed that adherence to a Mediterranean-style diet for an appropriate time significantly improved vascular health indices, including enhanced endothelial function, favorable alterations in lipid profiles and ceramide concentrations, and a reduction in inflammatory adipokines.

These findings support the diet’s potential as a holistic approach to mitigate cardiovascular risk factors and underscore its role in promoting overall cardiometabolic health.

The first aim of our study was indeed to was to evaluate the effects of adherence to a Mediterranean-style diet on some vascular health indices, such as endothelial function and arterial stiffness markers. We reported that at the six-month and twelve-month follow-ups, subjects in the Mediterranean Diet group experienced a more significant increase in RHI values than those in the control group.

Reactive hyperaemia is a well-established technique for the non-invasive assessment of peripheral microvascular function and a predictor of all-cause and cardiovascular morbidity and mortality.

Hypoxia stimulates vasodilation through mechanisms involving prostacyclin (PGI₂)-, adenosine-, nitric oxide (NO)-, and K⁺ channel-mediated hyperpolarization [27].

The reactive hyperaemia index (RHI) as measured by pulse amplitude tonometry (PAT), represents a simple, non-invasive measurement of endothelial function. Lower RHI has been correlated with cardiovascular (CV) risk factors, including obesity, total/HDL cholesterol ratio, diabetes, smoking and dyslipidaemia.

Endothelial and arterial stiffness indices have been defined as "surrogate" cardiovascular risk markers. The endothelium has been reported as an autocrine-paracrine organ. It has a crucial role in regulating tone and vascular structure in the control of humoral, nervous and mechanical inducements.

Our findings indicate that the adherence to a Mediterranean-style diet can correct endothelial dysfunction and increase mean RHI values.

A previous study [16] showed similar findings in patients at high cardiovascular risk. In the CORDIOPREV 805, patients with completed endothelial function study at baseline were randomized to follow a Mediterranean diet with endothelial function measurement repeated after 1 year. Patients who followed the Mediterranean diet had higher mean values of flow-mediated dilation (FMD) compared with those in the low-fat diet, even in those patients with severe endothelial dysfunction. These results suggest that the Mediterranean diet better modulates endothelial function compared with a low-fat diet and is associated with a better balance of vascular homeostasis in high cardiovascular risk and severe endothelial dysfunction.

The Mediterranean-style diet is rich in legumes, nuts, unrefined grains and fish, all significant L-arginine sources that sustains NO production by endothelium [30].

Multiple pathological conditions, such as hypertension, congestive heart failure, hypercholesterolemia, diabetes and metabolic syndrome are associated with the reduced bioavailability of NO [31–33]. Therefore, an increase in NO bioavailability could reduce the severity of these diseases [34–37].

Adherence to the Mediterranean-style diet has also been report to be able to have effects on the inflammatory state. The Mediterranean-style diet in comparison to a western meat diet is associated with a lower serum concentration of inflammatory markers [38].

The MOLI-SANI study [39] suggested that adherence to a Mediterranean-style diet reduces oxidative stress [40], vascular inflammation [39] and endothelial dysfunction [41]. Furthermore, other authors reported that lower serum concentration of VCAM-1, ICAM-1, E-selectin and P-selectin, CRP and IL-6 and a downregulation of adhesion molecules expressed on the surfaces of lymphocytes and monocytes after three months of implementation of a Mediterranean-style diet [42].

The second aim of our study was to analyse the effects of adherence to a Mediterranean-style diet on the lipidaemic profile in subjects at high cardiovascular risk. At the 6- and 12-month follow-ups, subjects treated with a Mediterranean-style diet showed lower mean serum total cholesterol levels, and lower mean serum LDL cholesterol than the control group.

Some studies have reported that moderate or high adherence to the Mediterranean-style diet can protect against an increased risk of atherosclerotic plaque formation and may provide protection against clinical vascular events.

Our study confirmed previous studies showing a favourable effect of adherence to a Mediterranean-style diet on the blood lipid profile [15,16,43].

The results of the study confirmed the impact of dietary factors on blood lipids. They also provided additional evidence on the response of high-density lipoprotein cholesterol to diet in free-living populations.

In our study, lipidaemic effects were observed at the 6-month follow-up and furtherly confirmed at the twelve-month follow-up.

Our study also aimed to evaluate the serum concentrations of the following ceramides: Cer 16, Cer-18, Cer-22 and Cer-24.

At 6 months, we report in the subjects of the Mediterranean Diet group lower values of Cer-24 and higher values of the Cer-22 and Cer-24/ Cer-16 ratio with statistically significant inter-group variability.

The same study group at the twelve-month follow-up also showed significantly lower values of C24:0 and C18, and higher values of the Cer-24/Cer-16 ratio.

Ceramides are bioactive lipids involved in cellular responses and apoptosis. Ceramide and its metabolites have been reported as a converging point between overeating and metabolic abnormalities that increase the risk of cardiometabolic diseases, such as insulin resistance and inflammation.

The intervention with the Mediterranean-style diet therefore modified the damaging effect of higher ceramide concentrations on CVD risk. There are two potential mechanisms for the modulatory effects of this diet on the ceramide pathway. First, the consumption of relevant dietary components can directly affect ceramide biosynthesis.

A study by Mantovani et al. [44] for example, evaluated how the plasma concentrations of ceramides Cer-16, Cer-18, Cer-22and Cer-24 were associated with the presence of inducible myocardial ischaemia, thus being independent positive predictors of defects of myocardial perfusion. Wang et al. [30] evaluated the concentrations of the ceramides mentioned above in a cohort of patients in the PREDIMED study and found that a greater risk of experiencing major cardiovascular events correlated with their increase.

These results suggest a positive association between baseline plasma ceramide levels and cardiovascular events and indicate how adherence to a Mediterranean-style diet can influence the potential negative relationship between elevated plasma ceramide concentrations and CVD.

Furthermore, our findings show an increase in the Cer-24/Cer-16 ratio, which appears to be inversely related to the risk of cardiovascular events, suggesting the utility of this ratio as a new biomarker of cardiovascular risk [45].

In our current study, we report that a higher degree of adherence to a Mediterranean-style diet induces a significant reduction in ceramide concentrations at the six-month and twelve-month follow-ups due a decrease in synthesis. This diet promotes an increase in the Cer-24/ Cer-16 ratio, which has a protective role against the risk of developing cardiovascular diseases.

Therefore, it is possible to hypothesise that greater adherence to a healthy lifestyle recommended by the Mediterranean Diet can improve the cardio- and cerebrovascular risk profile, which can be indirectly assessed by monitoring the ceramide ratio described above.

An additional objective of our research project was to evaluate the serum concentrations of inflammatory adipokines, correlating their change over follow-up visits with differences in the level of adherence to a Mediterranean-style dietary regimen, which may be related to an improvement in endothelial function as measured by RHI.

Our results showed that in subjects with a Mediterranean Diet adherence profile, compared to the control group in which a low-fat diet was prescribed, there was a significant reduction in the serum concentrations of resistin and visfatin, but an increase in the plasma levels adiponectin at the six-month follow-up. At the twelve-month follow-up, we observed a further, more significant reduction of resistin and visfatin serum levels in the Mediterranean Diet group, but a reduction of adiponectin serum levels in comparison to the levels at T1.

Human resistin plays a crucial regulatory role in the inflammatory response [46]. Resistin enhances the production of pro-inflammatory cytokines such as IL-12, IL-6, TNF-α and monocyte chemoattractant protein-1 (MCP-1) in PBMCs, macrophages and hepatic stellate cells through nuclear factor-κB (NF-κB) [47–49]. Furthermore, resistin inhibits the protein that mediates endothelial nitric oxide synthesis through oxidative stress in human endothelial cells [47] promotes the formation of human macrophages, induces a prothrombotic phenotype in human endothelial cells [48–49] and induces platelet activation by increasing the expression of P-selectin [49]. These results suggest that human resistin may play an essential regulatory role in modulating interactions between endothelial cells, and pathogenesis and progression of atherosclerosis [48,50]. The literature findings are consistent with the results obtained from a study conducted by our research team [26] reporting that diabetic subjects with a diabetic foot had higher plasma levels of IL-6 and resistin, defined as possible determinants of increased cardiovascular risk, but lower plasma levels of adiponectin.

Visfatin is one of the main adipokines secreted by adipose tissue. The visfatin level increases significantly in people with obesity due to increased BMI. Visfatin regulates immune cell trafficking and induces a chronic inflammatory state in adipocytes. It also induces insulin resistance [51]. Visfatin induces the formation of atherosclerotic regulating the extracellular matrix and angiogenesis degradation [51].

Current knowledge in this regard therefore suggests that an increase in visfatin and resistin levels contributes to the pathogenesis of endothelial dysfunction, which subsequently leads to a reduction in plasma concentrations of adiponectin [51]. This adipokine generally exerts an insulin-sensitizing, anti-inflammatory, and anti-apoptotic effect on many cell types and acts in the brain by increasing energy expenditure and inducing weight loss [52].

Considering the significant effect of the Mediterranean-style diet in reducing the levels of the adipokines mentioned above and increasing the synthesis of NO and adiponectin, it is likely that adherence to this dietary regimen can reduce the extent of endothelial dysfunction and, at the same time, keep the cardiovascular risk of these patients low.

These beneficial effects with regard of endothelial function evaluated by RHI and ISX and lipidaemic and adipokine effects have been also sustained by the results of multivariable regression analysis concerning the beneficial effects of education to the adherence to a Mediterranean Diet style.

The findings of this prospective study concerning the observed positive effects of adherence to a Mediterranean-style diet on adipokine serum levels, ceramide serum levels and an index of vascular health, such as RHI, may help to explain the cardiovascular and cerebrovascular protective relationship reported in previous studies from our own group with regard to ischaemic stroke, in particular, the atherosclerotic subtype [23] hemorrhagic cerebrovascular disorders[22] and congestive heart failure [21,53].

Conclusions

Our findings reported the possible strict relationship between lipidaemic and beneficial vascular effects of Mediterranean diet underlying the pathogenetic interplay of some adipokines such as visfatin and resistin and of some ceramide serum levels as possible lipidaemic cardiovascular markers.

The findings of our current study offer a further possible explanation with regard to the beneficial effects of a higher degree of adherence to a Mediterranean-style diet on multiple cardiovascular risk factors and the underlying mechanisms of atherosclerosis. Moreover, these findings provide an additional plausible interpretation of the results from observational and cohort studies linking high adherence to a Mediterranean-style diet with lower total mortality and a decrease in cardiovascular events and cardiovascular mortality.

Supporting information