Aging and Metabolic Reprogramming of Adipose-Derived Stem Cells Affect Molecular Mechanisms Related to Cardiovascular Diseases.

We performed a systematic search of the PubMed database for English-language articles related to the function of adipose-derived stem cells in the pathogenesis of cardiovascular diseases. In preclinical models, adipose-derived stem cells protected arteries and the heart from oxidative stress and inflammation and preserved angiogenesis. However, clinical trials did not reiterate successful treatments with these cells in preclinical models. The low success in patients may be due to aging and metabolic reprogramming associated with the loss of proliferation capacity and increased senescence of stem cells, loss of mitochondrial function, increased oxidative stress and inflammation, and adipogenesis with increased lipid deposition associated with the low potential to induce endothelial cell function and angiogenesis, cardiomyocyte survival, and restore heart function. Then, we identify noncoding RNAs that may be mechanistically related to these dysfunctions of human adipose-derived stem cells. In particular, a decrease in let-7, miR-17-92, miR-21, miR-145, and miR-221 led to the loss of their function with obesity, type 2 diabetes, oxidative stress, and inflammation. An increase in miR-34a, miR-486-5p, and mir-24-3p contributed to the loss of function, with a noteworthy increase in miR-34a with age. In contrast, miR-146a and miR-210 may protect stem cells. However, a systematic analysis of other noncoding RNAs in human adipose-derived stem cells is warranted. Overall, this review gives insight into modes to improve the functionality of human adipose-derived stem cells.

Noncoding RNAs Controlling Oxidative Stress in Cancer.

Mitochondria in cancer cells tend to overproduce reactive oxygen species (ROS), inducing a vicious cycle between mitochondria, ROS, genomic instability, and cancer development. The first part of this review deals with the role of noncoding RNAs in regulating mitochondrial ROS production and the expression of antioxidants in cancer cells, preventing the increase of ROS in the tumor microenvironment. In addition, cytotoxic T and natural killer cells release high levels of ROS, inducing cell death, while anti-immune regulatory T cells, tumor-associated M2 macrophages, and myeloid-derived suppressor cells, at least at the initial stage of tumor growth, release low levels of ROS supporting tumor growth. Therefore, this review's second part deals with noncoding RNAs' role in regulating the metabolic reprogramming of immune cells about ROS release. Furthermore, the enrichment of noncoding RNAs in microvesicles allows communication between cell types in a tumor and between a tumor and tumor-adjacent tissues. Therefore, the third part illustrates how noncoding RNA-containing microvesicles secreted by mesenchymal stem cells and primary tumor cells may primarily aid the shift of immune cells to a pro-oncogenic phenotype. Conversely, microvesicles released by tumor-adjacent tissues may have the opposite effect. Our review reveals that a specific noncoding RNA may affect oxidative stress by several mechanisms, which may have opposite effects on tumor growth. Furthermore, they may be involved in mechanisms other than regulating oxidative stress, which may level out their effects on oxidative stress and tumor growth. In addition, several noncoding RNAs might share a specific function, making it very unlikely that intervening with only one of these noncoding RNAs will block this particular mechanism. Overall, further validation of the interaction between noncoding RNAs about cancer types and stages of tumor development is warranted.

Oxidative Stress and Inflammation in Cardiovascular Diseases and Cancer: Role of Non-coding RNAs.

High oxidative stress, Th1/Th17 immune response, M1 macrophage inflammation, and cell death are associated with cardiovascular diseases. Controlled oxidative stress, Th2/Treg anti-tumor immune response, M2 macrophage inflammation, and survival are associated with cancer. MiR-21 protects against cardiovascular diseases but may induce tumor growth by retaining the anti-inflammatory M2 macrophage and Treg phenotypes and inhibiting apoptosis. Down-regulation of let-7, miR-1, miR-9, miR-16, miR-20a, miR-22a, miR-23a, miR-24a, miR-26a, miR-29, miR-30a, miR-34a, miR-124, miR-128, miR-130a, miR-133, miR-140, miR-143-145, miR-150, miR-153, miR-181a, miR-378, and miR-383 may aid cancer cells to escape from stresses. Upregulation of miR-146 and miR-223 may reduce anti-tumor immune response together with miR-21 that also protects against apoptosis. MiR-155 and silencing of let-7e, miR-125, and miR-126 increase anti-tumor immune response. MiR expression depends on oxidative stress, cytokines, MYC, and TGF-ß, and expression of silencing lncRNAs and circ-RNAs. However, one lncRNA or circ-RNA may have opposite effects by targeting several miRs. For example, PVT1 induces apoptosis by targeting miR-16a and miR-30a but inhibits apoptosis by silencing miR-17. In addition, levels of a non-coding RNA in a cell type depend not only on expression in that cell type but also on an exchange of microvesicles between cell types and tumors. Although we got more insight into the function of a growing number of individual non-coding RNAs, overall, we do not know enough how several of them interact in functional networks and how their expression changes at different stages of disease progression.

The marker of cobalamin deficiency, plasma methylmalonic acid, may help identifying lysosomal iron trapping in patients. Its possible utility for heart failure.

Iron deficiency is known to aggravate the prognosis of patients with heart failure. Iron has functions in the mitochondrial respiratory chain. In patients with reduced mitochondrial respiration, the mitochondrial ratio between the level of nicotinamide adenine dinucleotide and its reduced form decreases. Due to the mitochondrial-lysosomal interplay, decreased mitochondrial respiration also leads to inhibition of lysosomal hydrolysis. As a result, cobalamin and iron will be trapped in lysosomes. This will, even if iron and cobalamin have been consumed and absorbed in sufficient amounts, lead to their functional deficiencies.1 Functional iron deficiency can further impede mitochondrial respiration. Increased plasma levels of methylmalonic acid were shown to predict all-cause and cardiovascular mortality in the general population. Treatments targeting mitochondrial and lysosomal function may correct the functional deficiencies and improve prognosis in a subgroup of patients with heart failure, notably those with skeletal muscle wasting. Methylmalonic acid levels may be used for monitoring response to treatment, thereby identifying patients of the subgroup in which disease outcome may improve.

Mir-1, miR-122, miR-132, and miR-133 Are Related to Subclinical Aortic Atherosclerosis Associated with Metabolic Syndrome.

Previously, miR-1, miR-122, miR-126, miR-132, miR-133, and miR-370 were found to be related to coronary artery disease (CAD) progression. However, their relationship with subclinical atherosclerosis, especially in subjects with metabolic syndrome, is unknown. Therefore, our aim was to determine their relationship with arterial markers of subclinical atherosclerosis. Metabolic syndrome subjects (n = 182) with high cardiovascular risk but without overt cardiovascular disease (CVD) were recruited from the Lithuanian High Cardiovascular Risk (LitHiR) primary prevention program. The ardio-ankle vascular index (CAVI), augmentation index normalized to a heart rate of 75 bpm (AIxHR75), aortic pulse wave velocity (AoPWV), and carotid artery stiffness were assessed. MicroRNAs (miRs) were analyzed in serum. Pearson correlation and a univariate linear regression t-test showed that miR-1, miR-133b, and miR-133a were negatively associated with CAVI mean, whereas miR-122 was positively associated. MiR-1, miR-133b and miR-133a, and miR-145 were negatively associated with AIxHR75. MiR-122 correlated negatively with AoPWV. In multivariate linear regression models, miR-133b and miR-122 predicted CAVImean, miR-133 predicted AIxHR75, and miR-122 predicted AoPWV. MiR-132 predicted right carotid artery stiffness, and miR-1 predicted left carotid artery stiffness. The addition of smoking to miR-133b and miR-122 enhanced the prediction of CAVI. Age and triglycerides enhanced the prediction of AoPWV by miR-122. A cluster of four miRs are related to subclinical atherosclerosis in subjects with metabolic syndrome. Combined, they may have a more substantial diagnostic or prognostic value than any single miR. Future follow-up studies are needed to establish their clinical relevance.

Myeloid Krüppel-like factor 2 is a critical regulator of metabolic inflammation.

Substantial evidence implicates crosstalk between metabolic tissues and the immune system in the inception and progression of obesity. However, molecular regulators that orchestrate metaflammation both centrally and peripherally remains incompletely understood. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as an essential regulator of obesity and its sequelae. In mice and humans, consumption of a fatty diet downregulates myeloid KLF2 levels. Under basal conditions, myeloid-specific KLF2 knockout mice (K2KO) exhibit increased feeding and weight gain. High-fat diet (HFD) feeding further exacerbates the K2KO metabolic disease phenotype. Mechanistically, loss of myeloid KLF2 increases metaflammation in peripheral and central tissues. A combination of pair-feeding, bone marrow-transplant, and microglial ablation implicate central and peripheral contributions to K2KO-induced metabolic dysfunction observed. Finally, overexpression of myeloid KLF2 protects mice from HFD-induced obesity and insulin resistance. Together, these data establish myeloid KLF2 as a nodal regulator of central and peripheral metabolic inflammation in homeostasis and disease.

RNA-sequencing reveals that STRN, ZNF484 and WNK1 add to the value of mitochondrial MT-COI and COX10 as markers of unstable coronary artery disease.

Markers in monocytes, precursors of macrophages, which are related to CAD, are largely unknown. Therefore, we aimed to identify genes in monocytes predictive of a new ischemic event in patients with CAD and/or discriminate between stable CAD and acute coronary syndrome. We included 66 patients with stable CAD, of which 24 developed a new ischemic event, and 19 patients with ACS. Circulating CD14+ monocytes were isolated with magnetic beads. RNA sequencing analysis in monocytes of patients with (n = 13) versus without (n = 11) ischemic event at follow-up and in patients with ACS (n = 12) was validated with qPCR (n = 85). MT-COI, STRN and COX10 predicted new ischemic events in CAD patients (power for separation at 1% error rate of 0.97, 0.90 and 0.77 respectively). Low MT-COI and high STRN were also related to shorter time between blood sampling and event. COX10 and ZNF484 together with MT-COI, STRN and WNK1 separated ACS completely from stable CAD patients. RNA expressions in monocytes of MT-COI, COX10, STRN, WNK1 and ZNF484 were independent of cholesterol lowering and antiplatelet treatment. They were independent of troponin T, a marker of myocardial injury. But, COX10 and ZNF484 in human plaques correlated to plaque markers of M1 macrophage polarization, reflecting vascular injury. Expression of MT-COI, COX10, STRN and WNK1, but not that of ZNF484, PBMCs paired with that in monocytes. The prospective study of relation of MT-COI, COX10, STRN, WNK1 and ZNF484 with unstable CAD is warranted.

Single cell transcriptomics based-MacSpectrum reveals novel macrophage activation signatures in diseases.

Adipose tissue macrophages (ATM) are crucial for maintaining adipose tissue homeostasis and mediating obesity-induced metabolic abnormalities, including prediabetic conditions and type 2 diabetes mellitus. Despite their key functions in regulating adipose tissue metabolic and immunologic homeostasis under normal and obese conditions, a high-resolution transcriptome annotation system that can capture ATM multifaceted activation profiles has not yet been developed. This is primarily attributed to the complexity of their differentiation/activation process in adipose tissue and their diverse activation profiles in response to microenvironmental cues. Although the concept of multifaceted macrophage action is well-accepted, no current model precisely depicts their dynamically regulated in vivo features. To address this knowledge gap, we generated single-cell transcriptome data from primary bone marrow-derived macrophages under polarizing and non-polarizing conditions to develop new high-resolution algorithms. The outcome was creation of a two-index platform, MacSpectrum (https://macspectrum.uconn.edu), that enables comprehensive high-resolution mapping of macrophage activation states from diverse mixed cell populations. MacSpectrum captured dynamic transitions of macrophage subpopulations under both in vitro and in vivo conditions. Importantly, MacSpectrum revealed unique "signature" gene sets in ATMs and circulating monocytes that displayed significant correlation with BMI and homeostasis model assessment of insulin resistance (HOMA-IR) in obese human patients. Thus, MacSpectrum provides unprecedented resolution to decode macrophage heterogeneity and will open new areas of clinical translation.

Circular RNAs as Potential Theranostics in the Cardiovascular System.

Cardiovascular diseases (CVDs) represent the largest contributor to mortality worldwide. Identification of novel therapeutic targets and biomarkers for CVDs is urgently needed. Circular RNAs (circRNAs) are endogenous, abundant, and stable non-coding RNAs formed by back-splicing events. Their role as regulators of gene expression has been increasingly reported. Notably, circRNAs mediate essential physiological and pathological processes in the cardiovascular system. Our first aim, therefore, is to summarize recent advances in the role of circRNAs in cardiac development as well as in pathogenesis of various CVDs. Because circRNAs are stable in circulation and their dynamic changes may reflect different disease stages, they are considered ideal biomarkers. Therefore, our second aim is to review studies that have identified circulating circRNAs as biomarkers for CVDs. Finally, we discuss the shortage of functional studies and the limitations of available clinical studies and provide future perspectives.

Extracellular Vesicles in Cardiovascular Theranostics.

Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

Functional Role of Cardiovascular Exosomes in Myocardial Injury and Atherosclerosis.

Extracellular vesicles are now widely recognized as key players in the prevention, repair or progression of cardiovascular disease. Here we first focus on the functional roles of extracellular vesicles in the cross-talk between cardiomyocytes and endothelial cells, important for maintaining normal development and function of the heart. Second, we discuss the role of extracellular vesicles secreted by embryonic and non-embryonic stem cells in repairing cardiomyocyte function and in restoring angiogenic potential after myocardial ischemia-reperfusion injury. Third, we focus on the role of extracellular vesicles in Endothelial to Mesenchymal Transition (EndMT), leading to conversion of endothelial cells to fibroblasts, secretion of extracellular proteins collagen and fibronectin, and fibrosis. Finally, we discuss the role of extracellular vesicles secreted under stress by endothelial cells, macrophages and vascular smooth muscle cells in atherosclerosis. On aggregate, the reviewed preclinical studies present evidence that extracellular vesicles secreted by cardiomyocytes, fibroblasts, endothelial cells, immune-system-related cells, vascular smooth muscle cells and stem cells play an important role in the pathogenesis of cardiovascular disease. However, further studies are needed to gain better insight into the mechanisms used to select specific content to transfer to specific target cells, and to induce or repress signaling pathways in their target cells.

Low Cytochrome Oxidase 1 Links Mitochondrial Dysfunction to Atherosclerosis in Mice and Pigs.

BACKGROUND: Cytochrome oxidase IV complex regulates energy production in mitochondria. Therefore, we determined the relation of COX genes with atherosclerosis in mice and pigs. METHODS AND RESULTS: First, we compared atherosclerosis in the aortic arch of age-matched (24 weeks) C57BL/6J control (n = 10), LDL-receptor deficient (n = 8), leptin-deficient ob/ob (n = 10), and double knock-out (lacking LDL-receptor and leptin) mice (n = 12). Low aortic mitochondria-encoded cytochrome oxidase 1 in obese diabetic double knock-out mice was associated with a larger plaque area and higher propensity of M1 macrophages and oxidized LDL. Caloric restriction increased mitochondria-encoded cytochrome oxidase 1 and reduced plaque area and oxidized LDL. This was associated with a reduction of titer of anti-oxidized LDL antibodies, a proxy of systemic oxidative stress. Low of mitochondria-encoded cytochrome oxidase 1 was related to low expression of peroxisome proliferative activated receptors α, δ, and γ and of peroxisome proliferative activated receptor, gamma, co-activator 1 alpha reflecting mitochondrial dysfunction. Caloric restriction increased them. To investigate if there was a diabetic/obesity requirement for mitochondria-encoded cytochrome oxidase 1 to be down-regulated, we then studied atherosclerosis in LAD of hypercholesterolemic pigs (n = 37). Pigs at the end of the study were divided in three groups based on increasing LAD plaque complexity according to Stary (Stary I: n = 12; Stary II: n = 13; Stary III: n = 12). Low mitochondria-encoded cytochrome oxidase 1 in isolated plaque macrophages was associated with more complex coronary plaques and oxidized LDL. Nucleus-encoded cytochrome oxidase 4I1 and cytochrome oxidase 10 did not correlate with plaque complexity and oxidative stress. In mice and pigs, MT-COI was inversely related to insulin resistance. CONCLUSIONS: Low MT-COI is related to mitochondrial dysfunction, oxidative stress and atherosclerosis and plaque complexity.

Low MT-CO1 in Monocytes and Microvesicles Is Associated With Outcome in Patients With Coronary Artery Disease.

BACKGROUND: Cytochrome oxidase (COX) IV complex regulates energy production in mitochondria. Impaired COX gene expression is related to obesity and type 2 diabetes mellitus, but whether it is directly related to the incidence of cardiovascular events is unknown. We investigated whether COX gene expression in monocytes is predictive for cardiovascular events in coronary artery disease patients. To avoid monocyte isolation from fresh blood, we then aimed to validate our findings in monocyte-derived microvesicles isolated from plasma. METHODS AND RESULTS: We enrolled 142 consecutive patients undergoing diagnostic coronary angiography between June 2010 and January 2011 and followed 67 patients with stable coronary artery disease prospectively for at least 3 years. Twenty-two patients experienced a new cardiovascular event (32.8%). Circulating CD14+ monocytes and microvesicles were isolated with magnetic beads, and COX mRNA levels were measured with quantitative polymerase chain reaction, after normalization with 5 validated house-keeping genes. Patients in the lowest tertile of mitochondrial cytochrome oxidase, subunit I (MT-COI) in monocytes at baseline had a higher risk for developing a new event after adjusting for age, sex, (ex)smoking, body mass index, blood pressure, diabetes mellitus, low-density lipoprotein- and high-density lipoprotein-cholesterol, triglycerides, high-sensitivity C-reactive protein, interleukin-6, and number of diseased vessels (harzard ratio [HR], 3.95; 95% CI, 1.63-9.57). Patients in the lowest tertile of MT-COI in monocyte-specific microvesicles had also a higher risk of developing a new event (adjusted HR, 5.00; 95% CI, 1.77-14). CONCLUSIONS: In the current blinded study, low MT-COI in monocytes of coronary artery disease patients identifies a population at risk for new cardiovascular events. For the first time, we show that signatures in monocyte-specific microvesicles in plasma have similar predictive properties.

A Comparative Metabolomics Approach Reveals Early Biomarkers for Metabolic Response to Acute Myocardial Infarction.

Discovery of novel biomarkers is critical for early diagnosis of acute coronary syndrome (ACS). Serum metabolite profiling of ST-elevation myocardial infarction (STEMI), unstable angina (UA) and healthy controls was performed using gas chromatography mass spectrometry (GC/MS), solid-phase microextraction coupled to gas chromatography mass spectrometry (SPME-GC/MS) and nuclear magnetic resonance (1H-NMR). Multivariate data analysis revealed a metabolic signature that could robustly discriminate STEMI patients from both healthy controls and UA patients. This panel of biomarkers consisted of 19 metabolites identified in the serum of STEMI patients. One of the most intriguing biomarkers among these metabolites is hydrogen sulfide (H2S), an endogenous gasotransmitter with profound effect on the heart. Serum H2S absolute levels were further investigated using a quantitative double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). This highly sensitive immunoassay confirmed the elevation of serum H2S in STEMI patients. H2S level discriminated between UA and STEMI groups, providing an initial insight into serum-free H2S bioavailability during ACS. In conclusion, the current study provides a detailed map illustrating the most predominant altered metabolic pathways and the biochemical linkages among the biomarker metabolites identified in STEMI patients. Metabolomics analysis may yield novel predictive biomarkers that will potentially allow for an earlier medical intervention.

Identifying circulating microRNAs as biomarkers of cardiovascular disease: a systematic review.

The aim of the present study is to identify microRNAs (miRs) with high potential to be used as biomarkers in plasma and/or serum to clinically diagnose, or provide accurate prognosis for survival in, patients with atherosclerosis, coronary artery disease, and acute coronary syndrome (ACS). A systematic search of published original research yielded a total of 72 studies. After review of the risk of bias of the published studies, according to Cochrane Collaboration and the QUADUAS Group standards, 19 studies were selected. Overall 52 different miRs were reported. In particular, miR-133a/b (5 studies), miR-208a/b (6 studies), and miR-499 (7 studies) were well studied and found to be significant diagnostic and/or prognostic markers across different cardiovascular disease progression stages. miR-1 and miR-145b are potential biomarkers of ACS; miR-1 with higher sensitivity for all acute myocardial infarction (AMI), and miR-145 for STEMI and worse outcome of AMI. But when miRs were studied across different ACS study populations, patients had varying degrees of coronary stenosis, which was identified as an important confounder that limited the ability to quantitatively pool the study results. The identified miRs were found to regulate endothelial function and angiogenesis (miR-1, miR-133), vascular smooth muscle cell differentiation (miR-133, miR-145), communication between vascular smooth muscle and endothelial cell to stabilize plaques (miR-145), apoptosis (miR-1, miR-133, miR-499), cardiac myocyte differentiation (miR-1, miR-133, miR-145, miR-208, miR-499), and to repress cardiac hypertrophy (miR-133). Their role in these processes may be explained by regulation of shared RNA targets such as cyclin-dependent kinase inhibitor 1A (or p21), ETS proto-oncogene 1, fascin actin-bundling protein 1, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4, insulin-like growth factor 1 receptor LIM and SH3 protein 1, purine nucleoside phosphorylase, and transgelin 2. These mechanistic data further support the clinical relevance of the identified miRs. miR-1, miR-133a/b, miR-145, miR-208a/b, and miR-499(a) in plasma and/or serum show some potential for diagnosis of cardiovascular disease. However, biased selection of miRs in most studies and unexplained contrasting results are major limitations of current miR research. Inconsistencies need to be addressed in order to definitively identify clinically useful miRs. Therefore, this paper presents important aspects to improve future miR research, including unbiased selection of miRs, standardization/normalization of reference miRs, adjustment for patient comorbidities and medication, and robust protocols of data-sharing plans that could prevent selective publication and selective reporting of miR research outcomes.

Exosomes: emerging roles in communication between blood cells and vascular tissues during atherosclerosis.

PURPOSE OF REVIEW: Microvesicles, in general, and exosomes together with their delivered content in particular, are now being widely recognized as key players in atherosclerosis. We have previously reviewed the role of microvesicles in atherosclerosis pathogenesis, diagnosis and therapy. Here, we focus on the roles of exosomes and discuss their emergent role in mediating activation and response to inflammation, vessel infiltration and induction of coagulation. We will finally give an outlook to discuss novel detection techniques and systems biology based data analyses to investigate exosome-mediated cell-to-cell communication. RECENT FINDINGS: Recent research points to a role of exosomes in delivering apoptotic and inflammatory content between blood cells and vascular cells, with a potential contribution of exosomes secreted by adipose tissue. An atheroprotective role of exosomes in response to coagulation that may contrast with the procoagulatory role of platelet-derived larger microvesicles is envisaged. New detection and separation methods and systems biology techniques are emerging. CONCLUSION: We project that the development of novel detection, separation and analysis mechanism and systems-based analysis methods will further unravel the paracrine and endocrine 'communication protocol' between cellular players in atherosclerosis, mediating inflammation, oxidative stress and apoptosis.

Stevia-derived compounds attenuate the toxic effects of ectopic lipid accumulation in the liver of obese mice: a transcriptomic and metabolomic study.

There is a close interaction between Type 2 Diabetes, obesity and liver disease. We have studied the effects of the two most abundant Stevia-derived steviol glycosides, stevioside and rebaudioside A, and their aglycol derivative steviol on liver steatosis and the hepatic effects of lipotoxicity using a mouse model of obesity and insulin resistance. We treated ob/ob and LDLR-double deficient mice with stevioside (10 mg⋅kg(-1)⋅day-1 p.o., n = 8), rebaudioside A (12 mg⋅kg(-1)⋅day-1 p.o., n = 8), or steviol (5 mg⋅kg(-1)⋅day(-1) p.o., n = 8). We determined their effects on liver steatosis and on the metabolic effects of lipotoxicity by histological analysis, and by combined gene-expression and metabolomic analyses. All compounds attenuated hepatic steatosis. This could be explained by improved glucose metabolism, fat catabolism, bile acid metabolism, and lipid storage and transport. We identified PPARs as important regulators and observed differences in effects on insulin resistance, inflammation and oxidative stress between Stevia-derived compounds. We conclude that Stevia-derived compounds reduce hepatic steatosis to a similar extent, despite differences in effects on glucose and lipid metabolism, and inflammation and oxidative stress. Thus our data show that liver toxicity can be reduced through several pathophysiological changes. Further identification of active metabolites and underlying mechanisms are warranted.

Biomechanical factors in atherosclerosis: mechanisms and clinical implications.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors-mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current 'state of the art' on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research.