Patrizia Guarini

Epigenetic control of 11 beta-hydroxysteroid dehydrogenase 2 gene promoter is related to human hypertension.

BACKGROUND Lower activity of 11 beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) classically induces hypertension by leading to an altered tetrahydrocortisol- versus tetrahydrocortisone-metabolites (THFs/THE) shuttle. Recent cell culture and animal studies suggest a role for promoter methylation, a major epigenetic feature of DNA, in regulation of HSD11B2 expression. Little is known, however, of human HSD11B2 epigenetic control and its relationship with the onset of hypertension. OBJECTIVE To explore the possible relevance of HSD11B2 promoter methylation, by examining human peripheral blood mononuclear cell (PBMC) DNA and urinary THFs/THE ratio as a biochemical indicator of 11beta-HSD2 activity, in blood pressure control. METHODS Twenty-five essential hypertensives and 32 subjects on prednisone therapy were analyzed, the latter to investigate 11beta-HSD2 function in the development of hypertension. RESULTS Elevated HSD11B2 promoter methylation was associated with hypertension developing in glucocorticoid-treated patients in parallel with a higher urinary THFs/THE ratio. Essential hypertensives with elevated urinary THFs/THE ratio also showed higher HSD11B2 promoter methylation. CONCLUSIONS These results show a clear link between the epigenetic regulation through repression of HSD11B2 in PBMC DNA and hypertension.

Cardiovascular epigenetics: From DNA methylation to microRNAs

Epigenetic phenomena are defined as heritable mechanisms that establish and maintain mitotically stable patterns of gene expression without modifying the base sequence of DNA. The major epigenetic features of mammalian cells include DNA methylation, post-translational histone modifications and RNA-based mechanisms including those controlled by small non-coding RNAs (miRNAs). The impact of epigenetic mechanisms in cardiovascular pathophysiology is now emerging as a major player in the interface between genotype to phenotype variability. This topic of research has strict implications on disease development and progression, and opens up possible novel preventive strategies in cardiovascular disease. An important aspect of epigenetic mechanisms is that they are potentially reversible and may be influenced by nutritional-environmental factors and through gene-environment interactions, all of which have an important role in complex, multifactorial diseases such as those affecting the cardiovascular system. Gene expression regulation through the interplay of DNA methylation and histone modifications is well-established, although the knowledge about the function of epigenetic signatures in cardiovascular disease is still largely unexplored. The study of epigenetic markers is, therefore, a very promising frontier of science which may aid in a deeper understanding of molecular mechanisms underlying the modulation of gene expression in the biomolecule pathways linked to cardiovascular diseases. This review focuses on up-to-date knowledge pertaining to the role of epigenetics, from DNA methylation to miRNAs, in major cardiovascular diseases such as ischemic heart disease, hypertension, heart failure and stroke.

The MTHFR 1298A>C Polymorphism and Genomic DNA Methylation in Human Lymphocytes

Methylenetetrahydrofolate reductase (MTHFR) balances the pool of folate coenzymes in one-carbon metabolism for DNA synthesis and methylation, both implicated in carcinogenesis. Epidemiologic studies have shown that two functional polymorphisms in MTHFR gene, 677C>T and 1298A>C, are related to increased cancer risk. We aimed to analyze lymphocyte DNA from 198 subjects to evaluate the MTHFR 1298A>C polymorphism and folate status affecting genomic DNA methylation as a possible mechanism underlying the relationship between MTHFR polymorphisms and cancer susceptibility. Carriers of the 1298AA wild-type genotype showed lower genomic DNA methylation compared with 1298AC or 1298CC genotypes [3.72 versus 8.59 or 6.79 ng 5-methyl-2′-deoxycytidine (5-mCyt)/μg DNA, P < 0.0001 and P = 0.007, respectively]. When DNA methylation was evaluated according to plasma folate status, only 1298AA with low folate levels revealed diminished DNA methylation (P < 0.0001). Moreover, when the two MTHFR polymorphisms were concomitantly evaluated at the low folate status, DNA methylation was reduced only in 1298AA/677TT compared with 1298AA/677CC (3.11 versus 7.29 ng 5-mCyt/μg DNA, P = 0.001) and 1298CC/677CC genotypes (3.11 versus 7.14 ng 5-mCyt/μg DNA, P = 0.004). However, the high prevalence of 677TT mutants within the 1298AA group (79%) and the similar biochemical features of 1298AA/677CC and 1298CC/677CC combined genotypes suggest that the gene-nutrient interaction affecting DNA methylation in 1298AA is mainly due to the coexistence of the 677TT genotype and that the 1298A>C polymorphism may convey its protective effect not through this interaction but through another pathway in one-carbon metabolism. Further mechanistic studies are warranted to investigate how single polymorphisms as well as MTHFR combined genotypes exert their effect on cancer susceptibility.

Urinary prostasin : A candidate marker of epithelial sodium channel activation in humans

Prostasin is a serine peptidase hypothesized to regulate epithelial sodium channel (ENaC) activity in animals or on in vitro cultured cells. We investigated whether urinary prostasin may be a candidate marker of ENaC activation in humans. We studied 10 healthy volunteers and 8 hypertensive patients with raised aldosterone-to-renin ratio before and after spironolactone or saline/Florinef suppression test, respectively. Four healthy subjects were also studied before and after saline. Urinary prostasin was evaluated by SDS-PAGE, 2D maps, and Western blotting. Every sample of normotensive individuals was compared with the corresponding sample of urine collected after spironolactone or saline; every sample of hypertensive patients was compared with the corresponding sample of urine collected after saline or Florinef. Prostasin was detectable in all subjects regardless of gender, dietary sodium intake, and spironolactone treatment. Spironolactone (100 mg) increased urinary Na+/K+ ratio and decreased urinary prostasin in normotensives in whom the renin/aldosterone axis was activated by a low Na+ intake, but it was ineffective in individuals with high Na+ intake. Saline infusion also reduced prostasin in normotensive subjects. In contrast, prostasin paradoxically increased in urine of patients affected by primary aldosteronism after volume expansion. By 2D immunoblotting, several protein isoforms were observed, some of them being overexpressed after inhibition tests in patients with primary aldosteronism. In addition to a “basal” aliquot of prostasin, constitutively released in human urine regardless of sodium balance and aldosterone activation, there exists a second “aldosterone-responsive” aliquot modulated by Na+ intake and potentially suitable as candidate marker of ENaC activation.

Delta-5 and Delta-6 Desaturases: Crucial Enzymes in Polyunsaturated Fatty Acid-Related Pathways with Pleiotropic Influences in Health and Disease

Polyunsaturated fatty acids (PUFA) play pleiotropic and crucial roles in biological systems. Both blood and tissue levels of PUFA are influenced not only by diet, but to a large extent also by genetic heritability. Delta-5 (D5D) and delta-6 desaturases (D6D), encoded respectively by FADS1 and FADS2 genes, are the rate-limiting enzymes for PUFA conversion and are recognized as main determinants of PUFA levels. Alterations of D5D/D6D activity have been associated with several diseases, from metabolic derangements to neuropsychiatric illnesses, from type 2 diabetes to cardiovascular disease, from inflammation to tumorigenesis. Similar results have been found by investigations on FADS1/FADS2 genotypes. Recent genome-wide association studies showed that FADS1/FADS2 genetic locus, beyond being the main determinant of PUFA, was strongly associated with plasma lipids and glucose metabolism. Other analyses suggested potential link between FADS1/FADS2 polymorphisms and cognitive development, immunological illnesses, and cardiovascular disease. Lessons from both animal models and rare disorders in humans further emphasized the key role of desaturases in health and disease. Remarkably, some of the above mentioned associations appear to be influenced by the environmental context/PUFA dietary intake, in particular the relative prevalence of ω-3 and ω-6 PUFA. In this narrative review we provide a summary of the evidences linking FADS1/FADS2 gene variants and D5D/D6D activities with various traits of human physiopathology. Moreover, we focus also on the potentially useful therapeutic application of D5D/D6D activity modulation, as suggested by anti-inflammatory and tumor-suppressing effects of D6D inhibition in mice models.

Global DNA Hypomethylation in Peripheral Blood Mononuclear Cells as a Biomarker of Cancer Risk

Background: Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. Methods: From an original sample-set of 753 male and female adults (ages 64.8 ± 7.3 years), PBMCs DNA methylation was measured in 68 subjects with history of cancer at time of enrollment and 62 who developed cancer during follow-up. Age- and sex-matched controls for prevalent and incident cancer cases (n = 68 and 58, respectively) were also selected. Global DNA methylation was assessed by liquid chromatography/mass spectrometry (LC/MS). Methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype and plasma folate concentrations were also determined for the known gene-nutrient interaction affecting DNA methylation. Results: Cancer subjects had significantly lower PBMCs-DNA methylation than controls [4.39 (95% confidence intervals (CI), 4.25–4.53) vs. 5.13 (95% CI, 5.03–5.21) %mCyt/(mCyt+Cyt); P < 0.0001]. A DNA methylation threshold of 4.74% clearly categorized patients with cancer from controls so that those with DNA methylation less than 4.74% showed an increased prevalence of cancer than those with higher levels (91.5% vs. 19%; P < 0.001). Subjects with cancer at follow-up had, already at enrollment, reduced DNA methylation as compared with controls [4.34 (95% CI, 4.24–4.51) vs. 5.08 (95% CI, 5.05–5.22) %mCyt/(mCyt+Cyt); P < 0.0001]. Moreover, MTHFR677C>T genotype and folate interact for determining DNA methylation, so that MTHFR677TT carriers with low folate had the lowest DNA methylation and concordantly showed a higher prevalence of cancer history (OR, 7.04; 95% CI, 1.52–32.63; P = 0.013). Conclusions: Genomic PBMCs-DNA methylation may be a useful epigenetic biomarker for early detection and cancer risk estimation. Impact: This study identifies a threshold for PBMCs-DNA methylation to detect cancer-affected from cancer-free subjects and an at-risk condition for cancer based on genomic DNA methylation and MTHFR677C>T-folate status. Cancer Epidemiol Biomarkers Prev; 22(3); 348–55. ©2012 AACR.

G20210A Prothrombin Gene Polymorphism and Prothrombin Activity in Subjects With or Without Angiographically Documented Coronary Artery Disease

Background—G20210A prothrombin mutation has been associated with high prothrombin levels and an increased risk of venous thrombosis. The role of this common polymorphism, as well as that of prothrombin levels, in determining the risk of arterial disease is still somewhat controversial. Methods and Results—We determined the prevalence of the G20210A mutation and prothrombin activity in 660 individuals, of whom 436 had angiographically documented severe coronary artery disease (CAD patients) and 224 had normal coronary angiography (CAD-free control subjects). Heterozygosity for the 20210A allele was found in 5.3% of the CAD patients versus 3.1% of the CAD-free subjects (P =0.21). Similarly, no statistically significant difference was found between CAD patients with or without previous myocardial infarction (4.5% versus 5.3%, respectively;P =0.73). The genotype-phenotype correlation study showed a significant influence of the 20210A allele on prothrombin activity, with higher levels in carriers compared with noncarriers (153.2% versus 122.2%, respectively;P <0.001). Prothrombin activity was significantly higher in CAD patients than in CAD-free subjects (132.8% versus 123.3%, respectively;P <0.005). By multiple logistic regression, prothrombin activity in the upper tertile of the control distribution was significantly associated with CAD compared with prothrombin activity in the lower tertile (adjusted odds ratio 1.86, 95% CI 1.01 to 3.4). Conclusions—In a population with a clear-cut definition of the phenotype, the G20210A prothrombin mutation was not significantly associated, per se, with either angiographically documented CAD or myocardial infarction, whereas it significantly influenced prothrombin activity. In our population, high prothrombin activity itself was independently associated with CAD but not with the presence or absence of previous myocardial infarction.

Effects induced by olive oil-rich diet on erythrocytes membrane lipids and sodium-potassium transports in postmenopausal hypertensive women

Since we have observed that mo-nounsaturated fatty acids (MUFA) enriched diet modifies red cell membrane lipids and cation transport systems in normotensive subjects, we similarly evaluated a group of hypertensive patients undergoing an analogous dietary modification. In a group of 18 moderately hypertensive women, the diet was supplemented for two months with olive oil (about 45 g/day), which replaced an equal amount of seasoning fats. Before and after this period, red cell fatty acid composition was evaluated by gas-chromatography in order to verify diet compliance: a significant increase in oleic acid was observed, while the content of saturated and polyunsaturated fatty acids remained unchanged. After olive oil, maximal rates of Na-K pump (5580±329 vs 6995±390, plt;0.001) and Na-K cotransport ( Na-COT 544±52 vs 877±46, plt;0.001: K-COT 790±76 vs 1176±66, plt;0.001), cell Na content (9.58+0.4 vs 10.61 ±0.6, plt;0.03) and passive permeability for Na (936±74 vs 1836±102, plt;0.001 ) rose significantly. Although the reduction in maximal rate of the Li-Na CT after olive oil was not significant, it was the only cation transport parameter being correlated with the variations of membrane lipids, namely negatively with UFA (r=-0.528, plt;0.05) and positively with SFA (r=0.482, plt;0.005). The change in maximal rate of Li-Na CT was also correlated with the variation of systolic and diastolic BP (r=0.50, plt;0.03). No changes in membrane lipid composition and ion transport systems were observed in a group of 13 control patients kept on usual diet over the same period. Thus, olive oil supplementation affects the lipid composition of the cell membrane in hypertension. This change is in turn associated with a modification of membrane transport activity; in addition a significant reduction of blood pressure is obtained.

Effect of fish oil supplementation on erythrocyte lipid pattern, malondialdehyde production and glutathione-peroxidase activity in psoriasis

Erythrocytes from psoriatic patients have a significant increase in polyunsaturated fatty acids (p less than 0.001) especially in arachidonic acid (p less than 0.001). Glutathione peroxidase activity, in both erythrocytes and platelets, was stimulated when compared with normal cells (p less than 0.001, less than 0.02, respectively) and the production of malondialdehyde was also increased in psoriasis (p less than 0.01). The level of plasma selenium was significantly reduced (52.80 vs 72.49 ng/ml; p less than 0.001). alpha-Tocopherol and retinol were both normal in plasma of psoriatics. After two months of fish oil supplementation, the erythrocyte lipid pattern was changed, eicosapentaenoic and dochesaenoic acids substituting the arachidonate in the membrane. A reduction in malondialdehyde (p less than 0.01), a prolongation of bleeding time (p less than 0.05) and a further stimulation of glutathione-peroxidase (p less than 0.001) in both erythrocytes and platelets was also found.

Promoter methylation in coagulation F7 gene influences plasma FVII concentrations and relates to coronary artery disease

Background Plasma factor VII concentrations (FVIIa), a marker of coronary artery disease (CAD) risk, are influenced by genetic markers at the promoter site: the A2 allele, due to a 10bp insertion at position −323, is a determinant of lower FVIIa concentrations and reduced CAD risk, while the −402A allele, due to a G>A substitution, confers increased transcriptional activity in vitro resulting in higher FVIIa. Transcriptional regulation of F7 by epigenetic features is, however, still unknown as is the inter-relationship of genetic and epigenetic modifications at the promoter site. Objective To investigate a possible epigenetic regulation of the F7 gene at the promoter region and its link with functional F7 polymorphisms at the same site. Methods and results F7 promoter methylation and its relation to F7 promoter polymorphisms in modulating FVIIa and CAD risk were evaluated by methyl-specific PCR and bisulfite sequencing techniques in 253 subjects, of whom 168 had CAD and 88 were CAD-free. Plasma FVIIa was inversely related to methylation in A1A1 and −402GG, that is in the absence of the rare A2 and −402A allele. The higher FVIIa paralleled the lower methylation in A1A1 compared to A2A2 (p=0.035), while no variation in methylation was associated with the different −402G>A genotypes. The modulation of methylation-induced FVIIa concentrations was observed only in A1A1 where the higher methylation resulting in lower FVIIa was prevalent within the CAD-free group compared to the CAD group (p=0.011). Conclusions Epigenetic regulation through methylation of F7 promoter is associated with CAD by affecting plasma FVIIa concentrations in A1A1 genotypes.