Cardiac fat adipocytes: An optimized protocol for isolation of ready-to-use mature adipocytes from human pericardial adipose tissue.

A better understanding of the pathophysiology of cardiac fat depots is crucial to describe their role in the development of cardiovascular diseases. To this end, we have developed a method to isolate mature fat cells from the pericardial adipose tissue (PAT), the most accessible cardiac fat depot during cardiac surgery. Using enzymatic isolation, we were able to successfully obtain mature fat cells together with the corresponding cells of the stromal vascular fraction (SVF). We subjected the PAT adipocytes to thorough morphological and molecular characterization, including detailed fatty acid profiling, and simultaneously investigated their reactivity to external stimuli. Our approach resulted in highly purified fat cells with sustained viability for up to 72 h after explantation. Remarkably, these adipocytes responded to multiple challenges, including pro-inflammatory and metabolic stimuli, indicating their potential to trigger a pro-inflammatory response and modulate endothelial cell behavior. Furthermore, we have created conditions to maintain whole PAT in culture and preserve their viability and reactivity to external stimuli. The efficiency of cell recovery combined with minimal dedifferentiation underscores the promise for future applications as a personalized tool for screening and assessing individual patient responses to drugs and supplements or nutraceuticals.

Nutraceutical Supplementation as a Potential Non-Drug Treatment for Fibromyalgia: Effects on Lipid Profile, Oxidative Status, and Quality of Life.

Fibromyalgia (FM) is a chronic syndrome of unknown etiology, although many studies point to inflammation, oxidative stress, and altered mitochondrial metabolism as some of the cornerstones of this disease. Despite its socioeconomic importance and due to the difficulties in diagnosis, there are no effective treatments. However, the use of non-drug treatments is increasingly becoming a recommended strategy. In this context, the effects of supplementation of FM patients with an olive (poly)phenol, vitamin C, and vitamin B preparation were investigated in this work, analyzing complete blood count, biochemical, lipid, and coagulation profiles, and inflammation and oxidation status in blood samples. To gain a better understanding of the molecular mechanisms and pathways involved in the etiology of FM, a proteomic study was also performed to investigate the mechanisms of action of the supplement. Our results show that the nutraceutical lowers the lipid profile, namely cholesterol, and improves the oxidative status of patients as well as their quality of life, suggesting that this product could be beneficial in the co-treatment of FM. ClinicalTrials.gov (ID: NCT06348537).

Hydroxytyrosol decreases EDNRA expression through epigenetic modification in colorectal cancer cells.

The Mediterranean diet (MD) is one of the healthiest ones and is associated with a lower incidence of cardiovascular and cerebrovascular diseases as well as cancer. Extra virgin olive oil (EVOO) is probably the most idiosyncratic component of this diet. EVOO has been attributed with many healthful effects, which may be due to its phenolic components, e.g. including hydroxytyrosol (HT). Recent studies suggest that EVOO and HT have molecular targets in human tissues and modulate epigenetic mechanisms. DNA methylation is one of the most studied epigenetic mechanisms and consists of the addition of a methyl group to the cytosines of the DNA chain. Given the purported health effects of EVOO (poly)phenols, we analyzed the changes induced by HT in DNA methylation, in a colorectal cancer cell line. Caco-2 cells were treated with HT for one week or with the demethylating agent 5'-azacytidine for 48 h. Global DNA methylation was assessed by ELISA. DNA bisulfitation was performed and Infinium Methylation EPIC BeadChips were used to analyze the specific methylation of CpG sites. We show an increase in global DNA methylation in Caco-2 cells after HT treatment, with a total of 32,141 differentially methylated (CpGs DMCpGs). Interestingly, our analyses revealed the endothelin receptor type A gene (EDNRA) as a possible molecular target of HT. In summary, we demonstrate that cellular supplementation with HT results in a specific methylome map and propose a potential gene target for HT.

Therapeutic potential of plant-derived extracellular vesicles as nanocarriers for exogenous miRNAs.

Cell-to-cell communication strategies include extracellular vesicles (EVs) in plants and animals. The bioactive molecules in a diet rich in vegetables and fruits are associated with disease-preventive effects. Plant-derived EVs (PDEVs) are biogenetically and morphologically comparable to mammalian EVs and transport bioactive molecules, including miRNAs. However, the biological functions of PDEVs are not fully understood, and standard isolation protocols are lacking. Here, PDEVs were isolated from four foods with a combination of ultracentrifugation and size exclusion chromatography, and evaluated as vehicles for enhanced transport of synthetic miRNAs. In addition, the role of food-derived EVs as carriers of dietary (poly)phenols and other secondary metabolites was investigated. EVs from broccoli, pomegranate, apple, and orange were efficiently isolated and characterized. In all four sources, 4 miRNA families were present in tissues and EVs. miRNAs present in broccoli and fruit-derived EVs showed a reduced RNase degradation and were ferried inside exposed cells. EVs transfected with a combination of ath-miR159a, ath-miR162a-3p, ath-miR166b-3p, and ath-miR396b-5p showed toxic effects on human cells, as did natural broccoli EVs alone. PDEVs transport trace amounts of phytochemicals, including flavonoids, anthocyanidins, phenolic acids, or glucosinolates. Thus, PDEVs can act as nanocarriers for functional miRNAs that could be used in RNA-based therapy.

Plant-Based Diets Reduce Blood Pressure: A Systematic Review of Recent Evidence.

PURPOSE OF REVIEW: Accumulating data on the consumption of plant-based diets and their impact on blood pressure indicate a consensus that plant-based diets are linked to reduced blood pressure. The suggested mechanisms of action are manifold, and, in this systematic review, we provide a summary of the most recent findings on plant-based diets and their impact on blood pressure, along with an analysis of the molecules accountable for the observed effects. RECENT FINDINGS: The overwhelming majority of intervention studies demonstrate that plant-based diets result in lower blood pressure readings when compared to diets that are based on animal products. The various mechanisms of action are being clarified. The data discussed in this systematic review allow us to conclude that plant-based diets are associated with lower blood pressure and overall better health outcomes (namely, on the cardiovascular system) when compared to animal-based diets. The mechanisms of action are being actively investigated and involve many macro- and micronutrients plentiful in plants and the dishes prepared with them.

The ultra-processed foods hypothesis: a product processed well beyond the basic ingredients in the package.

The NOVA classification of food items has become increasingly popular and is being used in several observational studies as well as in nutritional guidelines and recommendations. We propose that there is a need for this classification and its use in the formulation of public health policies to be critically discussed and re-appraised. The terms 'processing' and 'ultra-processing', which are crucial to the NOVA classification, are ill-defined, as no scientific, measurable or precise reference parameters exist for them. Likewise, the theoretical grounds of the NOVA classification are unclear and inaccurate. Overall, the NOVA classification conflicts with the classic, evidence-based evaluation of foods based on composition and portion size because NOVA postulates that the food itself (or how much of it is eaten) is unimportant, but rather that dietary effects are due to how the food is produced. We contend that the NOVA system suffers from a lack of biological plausibility so the assertion that ultra-processed foods are intrinsically unhealthful is largely unproven, and needs further examination and elaboration.

Dietary Fatty Acids and Inflammation: Focus on the n-6 Series.

Among the polyunsaturated fatty acids (PUFAs), those belonging to the n-3 (or ω3) series, i.e., alpha-linolenic (ALA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids have been studied for decades from a pharma-nutritional viewpoint, namely in relation to cardiovascular health. More recent research is focusing on n-6 PUFAs, e.g., linoleic acid (LA), whose levels of consumption are much higher than those of n-3 and that cannot be used "pharmacologically". Perhaps because of this, the biological actions of n-6 PUFAs have not been investigated in details as those of their n-3 counterparts. However, an increasing body of evidence underscores their healthful actions on the cardiovascular system. Among the critiques to n-6 PUFAs and, particularly, LA there is the fact that they are precursors of pro-inflammatory eicosanoids. Hence, the hypothesis posits that we should reduce their intakes precisely to avoid increasing systemic, low-grade inflammation, i.e., one of the major etiological agents in degenerative diseases. In this narrative review, we address the issue of whether n-6 PUFAs are indeed pro-inflammatory, we discuss the most recent evidence of their role(s) in human health and prognosis, and we conclude that adequate intakes of n-6 fatty acids are associated with better cardiovascular health and child development.

Therapeutic potential of broccoli-derived extracellular vesicles as nanocarriers of exogenous miRNAs.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The wide-ranging biological activities of microRNAs stimulated research on disease mechanisms and is suggesting appealing therapeutic applications. When unprotected, miRNAs suffer from rapid degradation and appropriate strategies need to be developed to improve their therapeutic potential. Since the first observation of miRNAs being naturally transported by extracellular vesicles (EVs), the latter have been proposed as specific transport means for drug delivery, conferring stability and increasing resistance against RNase degradation. However, a standard, reproducible, and cost-effective protocol for EV isolation is lacking. Here, the use of broccoli-derived EVs as a therapeutic vehicle for extracellular RNA drug delivery was assessed. EVs were isolated from broccoli, combining ultracentrifugation and size exclusion chromatography methodology. Caco-2 cells were exposed to isolated EVs loaded with exogenous miRNAs and cellular viability was tested. The miRNAs were taken up by this intestinal cell line. Our results show that broccoli EVs can be efficiently isolated, characterized, and loaded with exogenous miRNAs, leading to toxicity in caco-2 cells. Because the pharmaceutical industry is searching for novel drug delivery nanovesicles with intrinsic properties such as low immunogenicity, stability to the gastrointestinal tract, ability to overcome biological barriers, large-scale production, cost-effectiveness, etc., broccoli-isolated nanovesicles might be suitable candidates for future pharmacological applications. We propose broccoli as a natural source of EVs, which are capable of transporting exogenous miRNAs with potential therapeutic effects and suggest that appropriate toxicological and randomized controlled trials as well as patent applications are warranted.

Hydroxytyrosol improves mitochondrial energetics of a cellular model of Alzheimer's disease.

Mitochondrial energetic deficit is one of the hallmarks of neurodegenerative disorders, e.g. Alzheimer´s disease (AD). Adherence to a Mediterranean diet is associated with lower incidence of cognitive decline and AD and extra virgin olive oil's (poly)phenols such as oleuropein and hydroxytyrosol (HT) are being actively studied in this respect. In this study, we assessed the effects of HT on mitochondrial energetic dysfunction in the 7PA2 cells cellular model, i.e. one of the best cellular models of Aß toxicity with a well-characterized mitochondrial dysfunction typically observed in AD. We report an increase of new mitochondria at 8 h post HT-treatment, which was followed by higher mitochondrial fusion. Further, ATP concentrations were significantly increased after 24 h of treatment with HT as compared with controls. Our data suggest that HT may revert the energetic deficit of a cellular model of AD by potentiating mitochondrial activity. Because HT is being proposed as dietary supplement or component of functional foods, future studies in appropriate animal models and - eventually - humans are warranted to further investigate its potential neuroprotective actions in AD.

Molecular and metabolic effects of extra-virgin olive oil on the cardiovascular gene signature in rodents.

BACKGROUND AND AIMS: Overweight and obesity are major risk factors for degenerative diseases, including cardiometabolic disorders and cancer. Research on fat and fatty acids' type is attracting less attention than that on carbohydrates. High adherence to a Mediterranean diet is associated with a better prognosis. One characteristic of the Mediterranean diet is extra-virgin olive oil (EVOO) as the foremost source of dietary fat. EVOO is different from other vegetable oils because it contains peculiar "minor" components, mainly phenolic in nature. Even though olive oil is highly caloric, unrestricted use of olive oil in the PREDIMED trial did not result in weight gain. We sought to study the effects of EVOO in an appropriate mouse model of increased body weight. Furthermore, we explored the biochemical and metabolomic responses to EVOO consumption. METHODS AND RESULTS: C57BL/6N male mice were weight-matched and fed ad libitum with the following diets, for 16 weeks: 1) saturated fatty acid diet (SFA) or 2) extra-virgin olive oil diet (EVOO), a custom-prepared diet, isocaloric compared to SFA, in which 82% of fat was replaced by high (poly)phenol EVOO. We evaluated glucose homeostasis, serum biochemistry and plasma metabolomics, in addition to cardiac and hepatic gene profile, and mitochondrial respiration rate. CONCLUSION: Replacing saturated fatty acids (e.g. lard) with EVOO translates into moderate yet beneficial cardiometabolic and hepatic effects. Future research will further clarify the mechanisms of action of EVOO (poly)phenols and their role in a balanced diet.

Prevention and Treatment of Atherosclerosis: The Use of Nutraceuticals and Functional Foods.

Nutritional interventions are effective and - in theory - easy to implement primary and secondary prevention strategies that reduce several risk factors of atherosclerosis and cardiovascular disease (CVD). Yet, because of (a) the severe impact of CVD in terms of mortality, morbidity, quality of life, and economy, (b) the proved role of LDL plasma concentrations as the most critical risk factor, and (c) the obstacles found both in terms of biological effects and compliance of the patient by an exclusively dietary intervention, food supplements or nutraceuticals are now valuable resources for physicians. As regards cholesterol control, several preparations are available in the market, and we will critically review them in this chapter.

Nutrition and health or nutrients and health?

Diet is an important contributor to human health and public health bodies are issuing guidelines aimed at favouring healthy food choices. The aim of our paper is to discuss the aspects underlying the concept of nutrient profiles, that is, defining levels of energy, some macronutrients, or salt which should not be exceeded in individual foods, according to the available evidence, to help in understanding to what extent such approach may actually be useful for improving nutrition and quality of life of European consumers. We list several pitfalls and oversimplifications of the current approaches to nutrient profiling and of the dichotomic classification of foods into "healthy" and "unhealthy" products. In view of the current "Facilitating healthier food choices - establishing nutrient profiles" EU initiative, we believe that further debate among all stakeholders is warranted and must consider all the limitations outlined in this paper.

Educating health care professionals on the importance of proper diets. An online course on nutrition, health, and sustainability.

The majority of university curricula for health professionals does not incorporate courses on human nutrition and its links with human and planetary health. This primarily applies to medical and pharmacy students, who have important counselling roles and are at the forefront of public health. To address this important issue, EIT Food recently launched an online course on nutrition, health, and sustainability. Learners were able to provide feedback on the course through an end-of-course survey and social interaction on the FutureLearn platform. The course was very well attended worldwide and received positive feedback from learners. A total of 3,858 students enrolled in the program, from >20 countries. Learners reported inadequate training on nutrition in their own curriculum and indicated they would use key insights from the course to inform their own practice. This report provides insights from the course, which could be used as guidance for future initiatives.

Orthorexia nervosa and dieting in a non-clinical sample: a prospective study.

PURPOSE: Orthorexia Nervosa (ON) is characterised by excessive attention to a dietary regimen perceived as healthy. A critical factor in the distinction between ON and other eating disorders (EDs) is the dichotomy of quality-versus-quantity of food intake. We investigated whether specific types of diet or dieting frequency are associated with orthorexic features, explored the overlap between ON and EDs symptoms, and examined which constructs are predictive of ON after 6 months. METHODS: A total of 1075 students (75.1% female, mean age 20.9) completed a set of questionnaires assessing Orthorexia, Eating Disorders, Obsessions and Compulsions, Anxiety and Depression; 358 individuals (79.9 female, mean age 20.9) agreed to participate in the study and completed the same questionnaires after 6 months. Different regression models were defined to investigate our hypothesis. RESULTS: Findings suggest that ON is associated with the number and type of diets followed over a lifetime. Moreover, participants with EDs, body dissatisfaction, or a dysfunctional idea of thinness are more likely to report a greater degree of ON features. After 6 months, the best predictors of ON characteristics are the same ON characteristics assessed at the first administration, with a significant role in the ideal of thinness. CONCLUSIONS: ON is more frequent in individuals with a previous diagnosis of EDs and in individuals who followed a restrictive diet or a vegan/vegetarian one; the number of lifetime diets, beliefs, and behaviors related to the ideals of thinness or body dissatisfaction is common features of ON. Moreover, considering that having ON features in the past is the best ON predictor in the present, we can presume that ON is a construct stable over time. LEVEL OF EVIDENCE: Level IV: Evidence obtained from multiple time series analysis such as case studies. (NB: Dramatic results in uncontrolled trials might also be regarded as this type of evidence).

Science-based policy: targeted nutrition for all ages and the role of bioactives.

Globally, there has been a marked increase in longevity, but it is also apparent that significant inequalities remain, especially the inequality related to insufficient 'health' to enjoy or at least survive those later years. The major causes include lack of access to proper nutrition and healthcare services, and often the basic information to make the personal decisions related to diet and healthcare options and opportunities. Proper nutrition can be the best predictor of a long healthy life expectancy and, conversely, when inadequate and/or improper a prognosticator of a sharply curtailed expectancy. There is a dichotomy in both developed and developing countries as their populations are experiencing the phenomenon of being 'over fed and under nourished', i.e., caloric/energy excess and lack of essential nutrients, leading to health deficiencies, skyrocketing global obesity rates, excess chronic diseases, and premature mortality. There is need for new and/or innovative approaches to promoting health as individuals' age, and for public health programs to be a proactive blessing and not an archaic status quo 'eat your vegetables' mandate. A framework for progress has been proposed and published by the World Health Organization in their Global Strategy and Action Plan on Ageing and Health (WHO (2017) Advancing the right to health: the vital role of law. https://apps.who.int/iris/bitstream/handle/10665/252815/9789241511384-eng.pdf?sequence=1&isAllowed=y . Accessed 07 Jun 2021; WHO (2020a) What is Health Promotion. www.who.int/healthpromotion/fact-sheet/en/ . Accessed 07 Jun 2021; WHO (2020b) NCD mortality and morbidity. www.who.int/gho/ncd/mortality_morbidity/en/ . Accessed 07 Jun 2021). Couple this WHO mandate with current academic research into the processes of ageing, and the ingredients or regimens that have shown benefit and/or promise of such benefits. Now is the time for public health policy to 'not let the perfect be the enemy of the good,' but to progressively make health-promoting nutrition recommendations.

Supplementation with alpha-linolenic acid and inflammation: a feasibility trial.

Consumption of omega-3 fatty acids, including the precursor α-linolenic acid (ALA) is often sub-optimal and not in line with international guidelines. Supplementation is debatable, but some individuals, e.g., pre-diabetic, low-grade inflammation, cardiometabolic yet otherwise healthy subjects, might benefit from supra-physiological omega-3 intake, particularly to lessen inflammation. We explored the feasibility of a large clinical trial by performing a pilot study to evaluate adherence, palatability, and self-reported side effects of ALA administration in a group of volunteers. We enrolled 12 individuals with borderline dyslipidemia or overweight, treated with dietary advice according to international guidelines and who had insufficient intakes of essential fatty acids. Subjects were followed for nutritional counselling and were matched with appropriate controls. Patients were administered 6 g/day of ALA, for two months. We report the absence of side effects. such as fishy aftertaste and gastrointestinal distress, in addition to a slight decrease of C-reactive protein concentrations (Identifier: ISRCTN13118704).

Microbiota and cardiovascular disease risk: A scoping review.

An increasing number of studies suggests that the oral and the intestinal microbiota may indirectly or directly influence cardiovascular risk. In this regard, the microbiota could act by modifying compounds naturally present in food, both in a potentially atherogenic sense and in a protective sense; on the other hand, specific bacterial strains whose growth could also be facilitated by compounds of alimentary origin, i.e. prebiotics could instead play direct effects on atherogenesis. In other words, the microbiota-food relationship is a bi-directional one in which the latter modifies the former that, in return, produces metabolites with healthful or noxious effects. In this scoping review, we examine some of the microbiota-cardiovascular risk interactions that, in light of the available evidence, can be considered to already enjoy convincing scientific solidity. Notably, we focus on the oral and intestinal microbiota, where research is most active, and we propose some future cardio-preventive opportunities: one would be to develop and test compounds that can inhibit the formation of microbiota-derived noxious molecules. After the development of appropriate, reliable, and inexpensive screening tools for metabotypes, personalized diets can be implemented and pertinent supplements could be prescribed. The other therapeutic and preventive route that could be traveled is that of microbiota modification, via the use of appropriate pro- and prebiotics.

Anti-inflammatory activity of argan oil and its minor components.

Argan oil is thought to be the most expensive edible oil worldwide. It is difficult to produce and the argan tree only grows in a limited geographical area, notably Morocco and Algeria. Because it is produced by mechanical means, argan oil contains "minor" components that might be endowed with healthful effects. We investigated in vivo the anti-inflammatory activities of argan oil and its unsaponifiable fraction, using diclofenac as the control, in a carrageenan-induced rat model of inflammation. Rats were given different amounts of argan oil or its unsaponifiable fraction, by gavage. We report that argan oil and its "minor" components effectively lessen the inflammatory actions of carrageenan. Far from being "pharmacological" the actions of argan oil are comparable with those of diclofenac in the short, i.e. 4 h term. Sustained consumption of argan oil might, therefore, contribute to lessen the burden of degenerative diseases associated with higher inflammatory status.

Wine's Phenolic Compounds and Health: A Pythagorean View.

In support of the J curve that describes the association between wine consumption and all-cause mortality, researchers and the lay press often advocate the health benefits of (poly)phenol consumption via red wine intake and cite the vast amount of in vitro literature that would corroborate the hypothesis. Other researchers dismiss such evidence and call for total abstention. In this review, we take a skeptical, Pythagorean stance and we critically try to move the debate forward by pointing the readers to the many pitfalls of red wine (poly)phenol research, which we arbitrarily treat as if they were pharmacological agents. We conclude that, after 30 years of dedicated research and despite the considerable expenditure, we still lack solid, "pharmacological", human evidence to confirm wine (poly)phenols' biological actions. Future research will eventually clarify their activities and will back the current recommendations of responsibly drinking moderate amounts of wine with meals.