Bioactive Compounds Formulated in Phytosomes Administered as Complementary Therapy for Metabolic Disorders.

Metabolic disorders (MDs), including dyslipidemia, non-alcoholic fatty liver disease, diabetes mellitus, obesity and cardiovascular diseases are a significant threat to human health, despite the many therapies developed for their treatment. Different classes of bioactive compounds, such as polyphenols, flavonoids, alkaloids, and triterpenes have shown therapeutic potential in ameliorating various disorders. Most of these compounds present low bioavailability when administered orally, being rapidly metabolized in the digestive tract and liver which makes their metabolites less effective. Moreover, some of the bioactive compounds cannot fully exert their beneficial properties due to the low solubility and complex chemical structure which impede the passive diffusion through the intestinal cell membranes. To overcome these limitations, an innovative delivery system of phytosomes was developed. This review aims to highlight the scientific evidence proving the enhanced therapeutic benefits of the bioactive compounds formulated in phytosomes compared to the free compounds. The existing knowledge concerning the phytosomes' preparation, their characterization and bioavailability as well as the commercially available phytosomes with therapeutic potential to alleviate MDs are concisely depicted. This review brings arguments to encourage the use of phytosome formulation to diminish risk factors inducing MDs, or to treat the already installed diseases as complementary therapy to allopathic medication.

Recommendations for detection, validation, and evaluation of RNA editing events in cardiovascular and neurological/neurodegenerative diseases.

RNA editing, a common and potentially highly functional form of RNA modification, encompasses two different RNA modifications, namely adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) editing. As inosines are interpreted as guanosines by the cellular machinery, both A-to-I and C-to-U editing change the nucleotide sequence of the RNA. Editing events in coding sequences have the potential to change the amino acid sequence of proteins, whereas editing events in noncoding RNAs can, for example, affect microRNA target binding. With advancing RNA sequencing technology, more RNA editing events are being discovered, studied, and reported. However, RNA editing events are still often overlooked or discarded as sequence read quality defects. With this position paper, we aim to provide guidelines and recommendations for the detection, validation, and follow-up experiments to study RNA editing, taking examples from the fields of cardiovascular and brain disease. We discuss all steps, from sample collection, storage, and preparation, to different strategies for RNA sequencing and editing-sensitive data analysis strategies, to validation and follow-up experiments, as well as potential pitfalls and gaps in the available technologies. This paper may be used as an experimental guideline for RNA editing studies in any disease context.

Elevated Levels of Circulating lncRNAs LIPCAR and MALAT1 Predict an Unfavorable Outcome in Acute Coronary Syndrome Patients.

Coronary artery disease (CAD) is a leading cause of mortality worldwide. In this study, we aimed to assess the potential of plasma long non-coding RNAs (lncRNAs) LIPCAR and MALAT1 and microRNAs (miRNAs) miR-142-3p and miR-155-5p to discriminate unstable CAD patients from stable ones. 23 stable angina (SA), 21 unstable angina (UA), and 50 ST-segment elevation myocardial infarction (STEMI) patients were enrolled; their plasma was collected. ncRNA plasma levels were evaluated using RT-qPCR. All measured ncRNA levels were significantly increased in UA patients' plasma compared to SA patients' plasma and in STEMI-with major adverse cardiovascular event (MACE) patients' plasma vs. STEMI-without MACE patients' plasma. ROC analysis showed that increased levels of LIPCAR and MALAT1 were associated with UA, and the prognostic model improved with the addition of miR-155-5p levels. The assessed lncRNAs discriminated between hyperglycemic (HG) and normoglycemic (NG) UA patients, and they were associated with MACE incidence in STEMI patients; this prediction was improved by the addition of miR-142-3p levels to the ROC multivariate model. We propose LIPCAR and MALAT1 as effective diagnostic markers for vulnerable CAD, their association with HG in UA patients, and as robust predictors for unfavorable evolution of STEMI patients.

Formulation of Phytosomes with Extracts of Ginger Rhizomes and Rosehips with Improved Bioavailability, Antioxidant and Anti-Inflammatory Effects In Vivo.

The poor water solubility of natural antioxidants restricts their bioavailability and therapeutic use. We aimed to develop a new phytosome formulation with active compounds from extracts of ginger (GINex) and rosehips (ROSAex) designed to increase their bioavailability, antioxidant and anti-inflammatory properties. The phytosomes (PHYTOGINROSA-PGR) were prepared from freeze-dried GINex, ROSAex and phosphatidylcholine (PC) in different mass ratios using the thin-layer hydration method. PGR was characterized for structure, size, zeta potential, and encapsulation efficiency. Results showed that PGR comprises several different populations of particles, their size increasing with ROSAex concentration, having a zeta potential of ~-21mV. The encapsulation efficiency of 6-gingerol and ß-carotene was >80%. 31P NMR spectra showed that the shielding effect of the phosphorus atom in PC is proportional to the amount of ROSAex in PGR. PGR with a mass ratio GINex:ROSAex:PC-0.5:0.5:1 had the most effective antioxidant and anti-inflammatory effects in cultured human enterocytes. PGR-0.5:0.5:1 bioavailability and biodistribution were assessed in C57Bl/6J mice, and their antioxidant and anti-inflammatory effects were evaluated after administration by gavage to C57Bl/6J mice prior to LPS-induced systemic inflammation. Compared to extracts, PGR induced a 2.6-fold increase in 6-gingerol levels in plasma and over 40% in the liver and kidneys, in parallel with a 65% decrease in the stomach. PGR treatment of mice with systemic inflammation increased the sera antioxidant enzymes paraoxonase-1 and superoxide dismutase-2 and decreased the proinflammatory TNFα and IL-1ß levels in the liver and small intestine. No toxicity was induced by PGR either in vitro or in vivo. In conclusion, the phytosome formulation of GINex and ROSAex we developed resulted in stable complexes for oral administration with increased bioavailability, antioxidant and anti-inflammatory potential of their active compounds.

The science behind soft skills: Do's and Don'ts for early career researchers and beyond. A review paper from the EU-CardioRNA COST Action CA17129.

Soft skills are the elementary management, personal, and interpersonal abilities that are vital for an individual to be efficient at workplace or in their personal life. Each work place requires different set of soft skills. Thus, in addition to scientific/technical skills that are easier to access within a short time frame, several key soft skills are essential for the success of a researcher in today's international work environment. In this paper, the trainees and trainers of the EU-CardioRNA COST Action CA17129 training school on soft skills present basic and advanced soft skills for early career researchers. Here, we particularly emphasize on the importance of transferable and presentation skills, ethics, literature reading and reviewing, research protocol and grant writing, networking, and career opportunities for researchers. All these skills are vital but are often overlooked by some scholars. We also provide tips to ace in aforementioned skills that are crucial in a day-to-day life of early and late career researchers in academia and industry.

Mitochondrial DNA Together with miR-142-3p in Plasma Can Predict Unfavorable Outcomes in Patients after Acute Myocardial Infarction.

Myocardial infarction is one of the leading causes of death worldwide, despite numerous efforts to find efficient prognostic biomarkers and treatment targets. In the present study, we aimed to assess the potential of six microRNAs known to be involved in cardiovascular diseases, cell-free DNA (cfDNA), and mitochondrial DNA (mtDNA) circulating in plasma to be used as prognostic tools for the occurrence of unfavorable outcomes such as major adverse cardiovascular events (MACE) after acute ST-segment elevation myocardial infarction (STEMI). Fifty STEMI patients were enrolled and monitored for 6 months for the occurrence of MACE. Plasma was collected at three time points: upon admission to hospital (T0), at discharge from hospital (T1), and 6 months post-STEMI (T6). Plasma levels of miR-223-3p, miR-142-3p, miR-155-5p, miR-486-5p, miR-125a-5p, and miR-146a-5p, as well as of cfDNA and mtDNA, were measured by RT-qPCR. Results showed that the levels of all measured miRNAs, as well as of cfDNA and mtDNA, were the most increased at T1, compared to the other two time points. In the plasma of STEMI patients with MACE compared to those without MACE, we determined increased levels of miRNAs, cfDNA, and mtDNA at T1. Hence, we used the levels of all measured parameters at T1 for further statistical analysis. Statistical analysis demonstrated that all six miRNAs and cfDNA plus mtDNA levels, respectively, were associated with MACE. The minimal statistical model that could predict MACE in STEMI patients was the combination of mtDNA and miR-142-3p levels, as evidenced by ROC analysis (AUC = 0.97, p < 0.001). In conclusion, the increased plasma levels of mtDNA, along with miR-142-3p, could be used to predict unfavorable outcomes in STEMI patients.

miR-146a-5p, miR-223-3p and miR-142-3p as Potential Predictors of Major Adverse Cardiac Events in Young Patients with Acute ST Elevation Myocardial Infarction-Added Value over Left Ventricular Myocardial Work Indices.

Acute ST elevation myocardial infarction (STEMI) remains a leading cause of morbidity and mortality worldwide despite continuous advances in diagnostic, prognostic and therapeutic methods. Myocardial work (MW) indices and miRNAs have both emerged as potential prognostic markers in acute coronary syndromes in recent years. In this study we aim to assess the prognostic role of myocardial work indices and of a group of miRNAs in young patients with STEMI. We enrolled 50 young patients (<55 years) with STEMI who underwent primary PCI and 10 healthy age-matched controls. We performed standard 2D and 3D echocardiography; we also calculated left ventricular global longitudinal strain (GLS) and the derived myocardial work indices. Using RT-PCR we determined the plasmatic levels of six miRNAs: miR-223-3p, miR-142-3p, miR-146a-5p, miR-125a-5p, miR-486-5p and miR-155-5p. We assessed the occurrence of major adverse cardiac events (MACE) at up to one year after STEMI. Out of 50 patients, 18% experienced MACE at the one-year follow-up. In a Cox univariate logistic regression analysis, myocardial work indices were all significantly associated with MACE. The ROC analysis showed that GWI, GCW and GWE as a group have a better predictive value for MACE than each separately (AUC 0.951, p = 0.000). Patients with higher miRNAs values at baseline (miR-223-3p, miR-142-3p and miR-146a-5p) appear to have a higher probability of developing adverse events at 12 months of follow-up. ROC curves outlined for each variable confirmed their good predictive value (AUC = 0.832, p = 0.002 for miR-223-3p; AUC = 0.732, p = 0.031 for miR-142-3p and AUC = 0.848, p = 0.001 for miR-146a-5p); the group of three miRNAs also proved to have a better predictive value for MACE together than separately (AUC = 0.862). Moreover, adding each of the miRNAs (miR-233, miR-142-3p and miR-146a-5p) or all together over the myocardial work indices in the regression models improved their prognostic value. In conclusion, both myocardial work indices (GWI, GCW and GWE) and three miRNAs (miR-223-3p, miR-142-3p and miR-146a-5p) have the potential to be used as prognostic markers for adverse events after acute myocardial infarction. The combination of miRNAs and MW indices (measured at baseline) rather than each separately has very good predictive value for MACE in young STEMI patients (C-statistic 0.977).

Clusterin, paraoxonase 1, and myeloperoxidase alterations induce high-density lipoproteins dysfunction and contribute to peripheral artery disease; aggravation by type 2 diabetes mellitus.

Peripheral artery disease (PAD) is an atherosclerotic disorder affecting arteries of the lower limbs, the major risk factors including dyslipidemia and diabetes mellitus (DM). We aimed to identify alterations of the proteins in high-density lipoproteins (HDL) associated with HDL dysfunction in PAD patients. HDL2 and HDL3 were isolated from plasma of PAD patients with/without DM (PAD-DM/PAD) and healthy subjects (N). Apolipoprotein AI (ApoAI), ApoAII, ApoCIII, clusterin (CLU), paraoxonase 1 (PON1), myeloperoxidase (MPO), and ceruloplasmin (CP) were measured in HDL2 /HDL3 and plasma. Oxidation and glycation of the analyzed proteins were assessed as malondialdehyde-protein adducts (MDA) and advanced glycation end-products (AGE), respectively. The anti-inflammatory effect of HDL3 was estimated as its potential to reduce monocyte adhesion to tumor necrosis factor α-activated endothelial cells. We show that in PAD patients compared to N subjects: (i) HDL2 presented increased levels of MDA-PON1, AGE-PON1, AGE-ApoAI, ApoAII, ApoCIII, and CP levels, and decreased PON1 levels; (ii) HDL3 had increased levels of MDA- and AGE-CLU and -ApoAI, MDA-PON1, ApoCIII, CLU, MPO, CP, and reduced PON1 levels. All these alterations were exacerbated by DM. These changes were more pronounced in HDL3 , which had reduced anti-inflammatory potential in PAD and became pro-inflammatory in PAD-DM. In PAD patients' plasma, CLU levels and MPO specific activity increased, while PON1 specific activity decreased. In conclusion, HDL function is altered in PAD patients due to multiple modifications of associated proteins that are aggravated by DM. Plasma CLU, MPO, and PON1 could constitute indicators of HDL dysfunction and contribute to risk stratification in PAD patients.

MiR-223-3p levels in the plasma and atherosclerotic plaques are increased in aged patients with carotid artery stenosis; association with HDL-related proteins.

BACKGROUND: Cardiovascular diseases are still the main cause of death worldwide. Our aim was to analyse the link between miR-223-3p levels, dysfunctional HDL and the age of patients with carotid artery stenosis (CAS). METHODS AND RESULTS: Thirty-two CAS patients enrolled for endarterectomy were divided in 2 groups: aged over 65 years (n = 19) and under 65 years (n = 13). Plasma samples and atherosclerotic plaques from the carotid artery were collected from all patients. Plaque levels of miR-223-3p and its primary transcript (pri-miR-223) were assessed, together with Drosha, Dicer, apolipoprotein (apo)A-I, apoE and myeloperoxidase (MPO) gene expression. In the plasma and plaques, miR-223-3p expression levels were significantly increased in CAS patients over 65 years. Positive correlations between plaque miR-223-3p and pri-miR-223 levels with Drosha, apoA-I and MPO expression were observed. Significantly increased miR-223-3p levels in the plasma of CAS patients over 65 years were measured. Significant correlations between plasma miR-223-3p levels and HDL-related proteins were determined. The variance of plasma miR-223-3p levels was predicted significantly by the multiple regression models using either age, clinical variables, blood lipids or oxidative and inflammatory parameters. Receiver operator characteristic analysis revealed that plasma miR-223-3p levels and HDL-related proteins (MPO activity/apoA-I ratio, MPO specific activity) were correlated with advanced age. CONCLUSIONS: Taken together, these data suggest that plasma levels of miR-223-3p are independently associated with ageing in CAS patients and that, correlated with parameters associated with dysfunctional HDL, could predict the aggravation of CAS in elderly patients.

CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction.

Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were measured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC.

Increased miR-142 Levels in Plasma and Atherosclerotic Plaques from Peripheral Artery Disease Patients with Post-Surgery Cardiovascular Events.

There is an intensive effort to identify biomarkers to predict cardiovascular disease evolution. We aimed to determine the potential of microRNAs to predict the appearance of cardiovascular events (CVEs) in patients with peripheral artery disease (PAD) following femoral artery bypass surgery. Forty-seven PAD patients were enrolled and divided into two groups, without CVEs (n = 35) and with CVEs (n = 12), during 1 year follow-up. Intra-surgery atherosclerotic plaques from femoral arteries were collected and the levels of miR-142, miR-223, miR-155, and miR-92a of the primary transcripts of these microRNAs (pri-miRNAs), and gene expression of Drosha and Dicer were determined. Results showed that, in the plaques, miR-142, miR-223, and miR-155 expression levels were significantly increased in PAD patients with CVEs compared to those without CVEs. Positive correlations between these miRNAs and their pri-miRNAs levels and the Dicer/Drosha expression were observed. In the plasma of PAD patients with CVEs compared to those without CVEs, miR-223 and miR-142 were significantly increased. The multiple linear regression analyses revealed significant associations among several plasma lipids, oxidative and inflammatory parameters, and plasma miRNAs levels. Receiver operator characteristic (ROC) analysis disclosed that plasma miR-142 levels could be an independent predictor for CVEs in PAD patients. Functional bioinformatics analyses supported the role of these miRNAs in the regulation of biological processes associated with atherosclerosis. Taken together, these data suggest that plasma levels of miR-142, miR-223, miR-155, and miR-92a can significantly predict CVEs among PAD patients with good accuracy, and that plasma levels of miR-142 can be an independent biomarker to predict post-surgery CVEs development in PAD patients.

Regulation of microRNAs in high-fat diet induced hyperlipidemic hamsters.

Dyslipidemia is a documented risk factor for cardiovascular diseases and other metabolic disorders. Therefore, the analysis of hyperlipidemia (HL)-related miRNAs is a potential approach for achieving new prognostic markers in lipid-metabolism related diseases. We aimed to analyze specific distribution of miRNAs in different tissues from HL animals. Golden Syrian hamsters were fed either regular chow (NL) or high-fat diet (HL) for 12 weeks. Microarray miRNAs profiling was performed in liver, heart and small intestine and data analyzed by R-studio software. Functional enrichment bioinformatics analysis was performed using miRWalk and DAVID tools. We observed a dysregulation of miRNAs in HL tissues evidencing a discrete distribution in the heart-liver axis and three lipid metabolism-related miRNAs were identified: hsa-miR-223-3p, hsa-miR-21-5p, and hsa-miR-146a-5p. Expression levels of these miRNAs were increased in HL livers and hearts. Functional bioinformatics analysis showed involvement of these miRNAs in the regulation of biological processes altered in HL conditions such as lipid metabolic process, fat cell differentiation, regulation of smooth muscle cells and cardiac septum development. We identified a set of miRNAs dysregulated in different tissues of HFD-induced HL hamsters. These findings motivate further studies aiming to investigate novel molecular mechanisms of lipid metabolism and atherogenic HL.

Hyperlipidemia Determines Dysfunctional HDL Production and Impedes Cholesterol Efflux in the Small Intestine: Alleviation by Ginger Extract.

SCOPE: To assess the impact of ginger extract (GIN) in stimulating the production of quality HDL and the cholesterol efflux in the small intestine (SI), key processes in the management of hyperlipidemia (HL)-induced hepatic steatosis, and atherosclerosis. METHODS AND RESULTS: Three groups of hamsters are used: (i) N, fed standard diet, (ii) HL, fed high-fat diet for 21 weeks, and (iii) HL-GIN, HL treated with GIN for the last 5 weeks of diet. Apolipoprotein A-I (apoA-I), malondialdehyde-apoA-I (MDA-apoA-I), paraoxonase1 (PON1), and myeloperoxidase (MPO) are measured in plasma and SI. ATP-binding cassette A1 transporter (ABCA1), ABCG5/G8, liver X receptor α/ß (LXRα/ß), peroxisome proliferator-activated receptor γ (PPARγ), and sirtuin1 (SIRT1) are assessed in the SI. Results show that in HL plasma, GIN decreases MDA-apoA-I, MPO/PON1 ratio and increases HDL-cholesterol/total cholesterol. In HL-SI, GIN decreases MDA-apoA-I and MPO, increases ApoA-I, PON1, and ABCA1, and restores cholesterol efflux disturbed by HL (SIRT1-LXRα/ß-PPARγ-ABCG8). GIN administration is associated with the reduction of the aortic valves lipid-deposits. CONCLUSION: In HL conditions, GIN stimulates the functional HDL production by restoring apoA-I quality and quantity through inhibition of the oxidative stress, and increases cholesterol efflux in the SI. These effects are associated with the restoration of SIRT1-LXRα/ß-PPARγ pathway.

Zingiber officinale extract administration diminishes steroyl-CoA desaturase gene expression and activity in hyperlipidemic hamster liver by reducing the oxidative and endoplasmic reticulum stress.

BACKGROUND: Stearoyl CoA desaturases (SCD) are enzymes that convert saturated to monounsaturated fatty acids and have increased activity in hepatic steatosis. PURPOSE: We aimed to investigate the potential of ginger extract (GIN) to modulate the liver SCD1 expression and activity in hyperlipidemic (HL) conditions, in order to lower lipid accumulation in the steatotic liver. STUDY DESIGN/METHODS: Male Golden Syrian hamsters were divided in three groups: (i) fed with standard chow (N), (ii) fed with standard chow plus 3% cholesterol and 15% butter for 21 weeks (HL), (iii) HL treated with GIN (800 µg/kg body weight/day) in the last 5 weeks of fat diet (HL-GIN). Cholesterol (C), triglycerides (TG), non-esterified fatty acids (NEFA), SCD1 estimated activity (C16:1n7/C16:0; C18:1n9/C18:0) and gene expression, acetyl-CoA carboxylase (ACC), thiobarbituric acid reactive substances (TBARS), paraoxonase1 (PON1) and myeloperoxidase (MPO) were determined in the plasma and liver of all hamsters. We measured protein expression of endoplasmic reticulum stress (ERS) markers, gene and protein expression of liver X receptor α/ß (LXRα/ß), peroxisome proliferator-activated receptor γ (PPARγ), ATP-binding cassette sub-family G member 5/8 (ABCG5/G8) and 7α-hydroxylase1 (CYP7A1) in all hamsters' livers. RESULTS: In plasma, in HL-GIN versus HL hamsters, SCD1 estimated activity was lower (27%; 15%, p < 0.05), NEFA levels decreased by 91%, p < 0.001, while C and TG levels did not vary; the oxidative stress expressed as MPO and TBARS levels decreased (15%; 11%, p < 0.01), while PON1 protein increased (75%, p < 0.05). In the liver of HL-GIN versus HL, C, TG, NEFA, MPO and TBARS levels decreased (8-40%, p < 0.05) and PON1 protein levels increased (30%, p < 0.05), SCD1 estimated activity decreased (8%; 9%, p < 0.05), in parallel with the reduced gene expression of SCD1 and ACC (70-80%, p < 0.05). The protein expression of the ERS sensors decreased (30-65%, p < 0.05), while that of ABCG5/G8, CYP7A1, LXRα/ß and PPARγ increased in HL-GIN (20-30%, p < 0.05) versus HL liver. CONCLUSION: GIN reduces SCD1 estimated activity and expression, as well as the lipids accumulated in the livers of HL hamsters. This is achieved through a mechanism involving the decrease of the oxidative and ERS, and the enhancement of cholesterol efflux.