Redox Homeostasis in Cardiovascular Disease: The Role of Mitochondrial Sirtuins.

Cardiovascular disease (CVD) is still the leading cause of death worldwide. Despite successful advances in both pharmacological and lifestyle strategies to fight well-established risk factors, the burden of CVD is still increasing. Therefore, it is necessary to further deepen our knowledge of the pathogenesis of the disease for developing novel therapies to limit even more its related morbidity and mortality. Oxidative stress has been identified as a common trait of several manifestations of CVD and could be a promising target for innovative treatments. Mitochondria are a major source of oxidative stress and sirtuins are a family of enzymes that generate different post-translational protein modifications, thus regulating important cellular processes, including cell cycle, autophagy, gene expression, and others. In particular, three sirtuins, SIRT3, SIRT4, and SIRT5 are located within the mitochondrial matrix where they regulate energy production and antioxidant pathways. Therefore, these sirtuins are strongly involved in the balance between oxidant and antioxidant mechanisms. In this review, we summarize the activities of these sirtuins with a special focus on their role in the control of oxidative stress, in relation to energy metabolism, atherosclerosis, and CVD.

Epigenetic Hallmarks of Fetal Early Atherosclerotic Lesions in Humans.

Importance: Although increasingly strong evidence suggests a role of maternal total cholesterol and low-density lipoprotein cholesterol (LDLC) levels during pregnancy as a risk factor for atherosclerotic disease in the offspring, the underlying mechanisms need to be clarified for future clinical applications. Objective: To test whether epigenetic signatures characterize early fetal atherogenesis associated with maternal hypercholesterolemia and to provide a quantitative estimate of the contribution of maternal cholesterol level to fetal lesion size. Design, Setting, and Participants: This autopsy study analyzed 78 human fetal aorta autopsy samples from the Division of Human Pathology, Department of Advanced Biomedical Sciences, Federico II University of Naples, Naples, Italy. Maternal levels of total cholesterol, LDLC, high-density lipoprotein cholesterol (HDLC), triglycerides, and glucose and body mass index (BMI) were determined during hospitalization owing to spontaneous fetal death. Data were collected and immediately processed and analyzed to prevent degradation from January 1, 2011, through November 30, 2016. Main Outcomes and Measurements: Results of DNA methylation and messenger RNA levels of the following genes involved in cholesterol metabolism were assessed: superoxide dismutase 2 (SOD2), low-density lipoprotein receptor (LDLR), sterol regulatory element binding protein 2 (SREBP2), liver X receptor α (LXRα), and adenosine triphosphate-binding cassette transporter 1 (ABCA1). Results: Among the 78 fetal samples included in the analysis (59% male; mean [SD] fetal age, 25 [3] weeks), maternal cholesterol level explained a significant proportion of the fetal aortic lesion variance in multivariate analysis (61%; P = .001) independently by the effect of levels of HDLC, triglycerides, and glucose and BMI. Moreover, maternal total cholesterol and LDLC levels were positively associated with methylation of SREBP2 in fetal aortas (Pearson correlation, 0.488 and 0.503, respectively), whereas in univariate analysis, they were inversely correlated with SREBP2 messenger RNA levels in fetal aortas (Pearson correlation, -0.534 and -0.671, respectively). Epivariations of genes controlling cholesterol metabolism in cholesterol-treated human aortic endothelial cells were also observed. Conclusions and Relevance: The present study provides a stringent quantitative estimate of the magnitude of the association of maternal cholesterol levels during pregnancy with fetal aortic lesions and reveals the epigenetic response of fetal aortic SREBP2 to maternal cholesterol level. The role of maternal cholesterol level during pregnancy and epigenetic signature in offspring in cardiovascular primary prevention warrants further long-term causal relationship studies.

Epigenetics and type 1 diabetes: mechanisms and translational applications.

Type 1 diabetes (T1D) is an irreversible degenerative disease with severe complications such as heart disease, nephropathy, neuropathy, and retinopathy. Although exogenous insulin administration is a life-saving therapy, it does not cure the disease. This review addresses the epigenetic mechanisms responsible for the development of T1D and discusses epigenetic-based strategies for prevention and treatment of the disease. We describe novel epigenetic biomarkers for the identification of susceptible individuals and the establishment of innovative therapies with epidrugs and cell therapy to regenerate the lost ß-cells. Despite the wealth of promising data regarding the potential benefits of epigenetic tools to reduce the burden of T1D, clinical trials are still very few, and this issue needs to be resolved in the near future.

Possible Muscle Repair in the Human Cardiovascular System.

The regenerative potential of tissues and organs could promote survival, extended lifespan and healthy life in multicellular organisms. Niches of adult stemness are widely distributed and lead to the anatomical and functional regeneration of the damaged organ. Conversely, muscular regeneration in mammals, and humans in particular, is very limited and not a single piece of muscle can fully regrow after a severe injury. Therefore, muscle repair after myocardial infarction is still a chimera. Recently, it has been recognized that epigenetics could play a role in tissue regrowth since it guarantees the maintenance of cellular identity in differentiated cells and, therefore, the stability of organs and tissues. The removal of these locks can shift a specific cell identity back to the stem-like one. Given the gradual loss of tissue renewal potential in the course of evolution, in the last few years many different attempts to retrieve such potential by means of cell therapy approaches have been performed in experimental models. Here we review pathways and mechanisms involved in the in vivo repair of cardiovascular muscle tissues in humans. Moreover, we address the ongoing research on mammalian cardiac muscle repair based on adult stem cell transplantation and pro-regenerative factor delivery. This latter issue, involving genetic manipulations of adult cells, paves the way for developing possible therapeutic strategies in the field of cardiovascular muscle repair.

Polycomb YY1 is a critical interface between epigenetic code and miRNA machinery after exposure to hypoxia in malignancy.

Yin Yang 1 (YY1) is a member of polycomb protein family involved in epigenetic modifications and transcriptional controls. We have shown that YY1 acts as positive regulator of tumor growth and angiogenesis by interfering with the VEGFA network. Yet, the link between polycomb chromatin complex and hypoxia regulation of VEGFA is still poorly understood. Here, we establish that hypoxia impairs YY1 binding to VEGFA mRNA 3'UTR (p<0.001) in bone malignancy. Moreover, RNA immunoprecipitation reveals the formation of triplex nuclear complexes among YY1, VEGFA DNA, mRNA, and unreached about 200 fold primiRNA 200b and 200c via Dicer protein. In this complex, YY1 is necessary to maintain the steady-state level of VEGFA expression while its silencing increases VEGFA mRNA half-life at 4 h and impairs the maturation of miRNA 200b/c. Hypoxia promotes histone modification through ubiquitination both of YY1 and Dicer proteins. Hypoxia-mediated down-regulation of YY1 and Dicer changes post-transcriptional VEGFA regulation by resulting in the accumulation of primiRNA200b/c in comparison to mature miRNAs (p<0.001). Given the regulatory functions of VEGFA on cellular activities to promote neoangiogenesis, we conclude that YY1 acts as novel critical interface between epigenetic code and miRNAs machinery under chronic hypoxia in malignancy.

Current concepts in histocompatibility during heart transplant.

Sensitized candidates for heart transplant usually end up on a long waiting list and have an increased risk of rejection, graft loss, and incidence of cardiac allograft vasculopathy. An increasing number of studies have demonstrated the negative effect of preformed and posttransplant antibodies on graft survival. Thus, in sensitized patients, the combination of new, appropriate, desensitization protocols, and monitoring of posttransplant development of donor-specific antibodies may improve short-term and long-term outcomes. Introduction of more-sensitive and more-specific techniques for antibody detection provides a valid tool for assessing the degree of pretransplant HLA histocompatibility, and, therefore, predicting the results of crossmatch in sensitized patients, which are difficult to transplant. Currently, there are no accurate and standard methods to determine the functional characteristics of antibodies detected by solid-phase assay and, therefore, to predict their clinical relevance. Therefore, the future of heart transplantation requires a better understanding of tissue typing techniques and the effect of anti-HLA antibodies on clinical outcome to prevent discrimination against sensitized patients at the time of organ allocation.

cAMP and Pyk2 interact to regulate prostate cell proliferation and function.

In cultured prostate cancer cells cAMP blocks proliferation and induces neuroendocrine differentiation. Pyk2 expression inversely correlates with malignancy of prostate cancer. The aim of this study was to investigate the interaction between cAMP and Pyk2 in the prostate. EPN cells, a line derived from human normal prostate expressing Pyk2, and EPN-PKM3 cells, an EPN clone bearing a Pyk2 kinase-negative mutant, were adopted as model system. cAMP inhibited cell growth in both prostate cell lines, and activated Pyk2, but not ERK1/2, in EPN cells. cAMP treatment, abolished the activation of AKT1, an important component of the pro-survival pathway, in the EPN cells but not in EPN-PKM3 cells. Finally, upon cAMP treatment, EPN and EPN-PKM3 cells exhibited different expression patterns of HOX genes, an important network controlling cell identity. These data demonstrated for the first time that Pyk2 and cAMP interact in regulating prostate cell functions and in "keeping" prostate identity.

Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improves the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension.

BACKGROUND: Sulfhydryl angiotensin-converting enzyme (ACE) inhibitors exert antiatherosclerotic effects in preclinical models and antioxidant effects in patients. However, whether ACE inhibitors have any clinically significant antiatherogenic effects remains still debated. OBJECTIVES: In mildly hypertensive patients, we evaluated the effect of the sulfhydryl ACE inhibitor zofenopril in comparison with the carboxylic ACE inhibitor enalapril on carotid atherosclerosis (intima-media thickness [IMT] and vascular lumen diameter) and systemic oxidative stress (nitrite/nitrate, asymmetrical dimethyl-l-arginine, and isoprostanes). METHODS: In 2001, we started a small prospective randomized clinical trial on 48 newly diagnosed mildly hypertensive patients with no additional risk factors for atherosclerosis (eg, hyperlipidemia, smoke habit, familiar history of atherosclerosis-related diseases or diabetes). Patients were randomly assigned either to the enalapril (20 mg/d, n = 24) or the zofenopril group (30 mg/d, n = 24); the planned duration of the trial was 5 years. Carotid IMT and vascular lumen diameter were determined by ultrasonography for all patients at baseline and at 1, 3, and 5 years. Furthermore, nitrite/nitrate, asymmetrical dimethyl-l-arginine, and isoprostane levels were measured. RESULTS: In our conditions, IMT of the right and left common carotid arteries was similar at baseline in both groups (P = NS). Intima-media thickness measurements until 5 years revealed a significant reduction in the zofenopril group but not in the enalapril group (P < .05 vs enalapril-treated group). This effect was coupled with a favorable nitric oxide/oxidative stress profile in the zofenopril group. CONCLUSIONS: Long-term treatment with the sulfhydryl ACE inhibitor zofenopril besides its blood pressure-lowering effects may slow the progression of IMT of the carotid artery in newly diagnosed mildly hypertensive patients.

Antioxidants increase number of progenitor endothelial cells through multiple gene expression pathways.

To date, there is no report on the effect of antioxidants on endothelial progenitor cells (EPCs). This study shows that in vitro incubation of EPCs with vitamin C and E reverted the already well documented lowering effect of TNF-alpha on EPC number and increased p-p38 expression levels. In order to document major changes of gene expression levels and gain insight into signalling pathways, microarray analysis was performed and a significant variation of the expression of 5389 genes in EPCs following antioxidant treatment was detected. Also in vivo evidence is provided about the positive effect of antioxidant vitamins on EPCs, since vitamin C and E supplementation potentiated the physical training-induced increase of EPC number and VEGF levels. Together, these data indicate that antioxidant treatment ameliorates EPC number and causes major changes of gene expression within these cells in vitro. Furthermore, concomitant antioxidant supplementation and physical training in vivo raised the levels of circulating EPCs and serum VEGF more than physical training alone.

Therapeutic targeting of the stem cell niche in experimental hindlimb ischemia.

BACKGROUND: The custom microenvironment 'vascular niche' is a potential therapeutic target for several pathophysiological conditions. Osteoblasts regulate the hematopoietic stem cell niche, and activation of the parathyroid hormone (PTH) receptor can increase the number of cells mobilized into the bloodstream. METHODS: C57Bl/6 mice were randomly assigned treatment with granulocyte-colony stimulating factor (G-CSF), PTH, G-CSF plus PTH or saline. All mice underwent hindlimb ischemia. Blood flow was measured by laser Doppler imaging. Indices of capillary activity were determined by electron microscopy in muscle tissue. CD34(+) and Ki67(+) cells were detected and evaluated by immunofluorescence, apoptosis by TUNEL, surface antigen and endothelial progenitor cells by fluorescence-activated cell sorting analysis, and vascular endothelial growth factor-164 and angiopoietin-1 expression by reverse-transcriptase polymerase chain reaction. Frozen bone marrow sections were stained for antigen-specific B cells and fibronectin and analyzed by confocal laser scanning microscopy. RESULTS: Following mobilization induced by G-CSF treatment, mice also treated with PTH showed increases in blood flow, capillary density, nitrite/nitrate release, angiogenic factors and circulating progenitor cells, as well as reduced apoptosis, fibrosis, oxidative stress and inflammation in ischemic muscles. Furthermore, hematopoietic antigen-specific B cells in the bone marrow were also increased by G-CSF alone and in combination with PTH. CONCLUSIONS: PTH might increase the efficiency of hematopoietic stem-cell-based therapy in a recognized model of peripheral ischemia. Our translational experimental therapeutic targeting of the vascular niche points to novel clinical targets for the hematopoietic stem-cell treatment of ischemic vascular diseases.

The dietary antioxidant resveratrol affects redox changes of PPARalpha activity.

BACKGROUND AND AIMS: Gene-environment interaction is behind the pathogenesis of most widespread diseases, and nutrition is among the environmental factors with the highest impact on human health. The mechanisms involved in the interaction between nutritional factors and the genetic background of individuals are still unclear. The aim of this study was to investigate whether resveratrol (RES), an antioxidant polyphenol of red wine, can influence the activity of PPARalpha in the rat hepatoma cell line McArdle-RH7777. PPARalpha is a transcriptional factor that regulates gene expression when activated by endogenous or exogenous long-chain fatty acids. Its activation results in significant protection from cardiovascular diseases in humans. METHODS AND RESULTS: By means of the electromobility shift assay (EMSA), we observed that PPARalpha is redox-sensitive as it displays reduced DNA-binding activity following in vivo treatment of the cells with 1mmol/L diethylmaleate (DEM), a glutathione-depleting agent. This finding could be relevant considering the important role of redox balance in pathological and physiological processes. We also observed a dual effect of 100mumol/L RES on PPARalpha activity: it was able to prevent, to a large extent, the DEM-induced reduction of DNA-binding activity at earlier time points, when the effect of DEM was stronger, but it depressed PPARalpha activity at later time points, when the effect of DEM was greatly reduced. CONCLUSION: A nutritional substance, such as RES, is able to influence the activity of gene-regulating factors, but the net effect is difficult to predict when the compound involved has multiple biological properties. Caution is therefore warranted before drawing conclusions about the potential benefits of RES for human health.

Variants of uncoupling protein-2 gene and obesity: interaction with peroxisome proliferator-activated receptorgamma2.

OBJECTIVE: To analyse the association of the UCP2 gene, alone or in combination with the PPARgamma2 gene, with obesity. DESIGN: Cross-sectional, case-control study. STUDY POPULATION: From a working population of 4500 Italian Caucasian employees of the Italian telephone company participating in a firm-sponsored health screening programme, we selected all those with obesity [n = 122; body mass index (BMI) > or = 30 kg/m2]. For each case, three nonobese age- and sex-matched individuals were selected as controls from the same population (n = 374). Included in the study were also 76 severely obese (BMI > or = 40 kg/m2) patients consecutively admitted to the obesity clinic of the department. Diabetic individuals were excluded. MEASUREMENTS: The -866G/A UCP2 and the Pro12Ala PPARgamma2 polymorphisms were determined on genomic DNA of the studied individuals. Several metabolic and anthropometric measures were also obtained, like plasma glucose, insulin, triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol and BMI. RESULTS: BMI, plasma glucose, insulin, triglycerides, total and HDL cholesterol were not significantly different in carriers and noncarriers of the -866G/A variant. No significant association was observed between the -866G/A UCP2 gene polymorphism and moderate or severe obesity. This was also observed when the UCP2 polymorphism was analysed in combination with the PPARgamma2 polymorphisms. CONCLUSIONS: The -866G/A variants of the UCP2 gene are not associated with either obesity or other features of the metabolic syndrome in the studied groups of the Italian population. This negative finding is not modified after a combined analysis of the UCP2 polymorphism and the Pro12Ala polymorphism of PPARgamma2.

De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase-1 expression and fatty acid metabolism in liver of fenofibrate-treated rats.

Uncoupling protein 3 (UCP3) is a member of the mitochondrial carrier superfamily, preferentially expressed in skeletal muscle. Its function is not fully understood and it is debated whether it uncouples oxidative phosphorylation as does UCP1 in brown adipose tissue. Recent evidences suggest a role for UCP3 in the flux of fatty acids in and out mitochondria and their utilization in concert with mitochondrial thioesterase-1 (MTE-1). In fact, mice overexpressing muscle UCP3 also show high levels of MTE-1. Fenofibrate is a hypolipidemic drug that prevents body weight gain in diet-induced obese rats and enhances lipid metabolism by activating peroxisome proliferator-activated receptors (PPARs). Because fatty acids and fenofibrate stimulate PPARs and in turn UCP3, we investigated whether UCP3 expression might be induced 'de novo' in situations of increased hepatic mitochondrial fatty acid utilization caused by a combined effect of a high-fat diet and fenofibrate treatment. We also investigated whether Mte-1 expression and beta-oxidation were affected. We show here that Ucp3 is induced in liver of fenofibrate-treated rats at the mRNA and protein level. Expression was restricted to hepatocytes and was unevenly distributed in the liver. No increase in cell proliferation, inflammatory or fibrotic responses was found. Mte-1 expression and mitochondrial beta-oxidation were upregulated. Thus, Ucp3 can be transactivated in tissues where it is normally silent and fenofibrate can attain this effect in liver. The data demonstrate that UCP3 is involved in fatty acid utilization and support the notion that UCP3 and MTE-1 are linked within the same metabolic pathway.