Daniela Lucchesi

Oxidative stress in response to high glucose levels in endothelial cells and in endothelial progenitor cells: evidence for differential glutathione peroxidase-1 expression.

Endothelial cells and endothelial progenitor cells (EPCs) play a key role in the pathogenesis of vascular disease. Both cell types are affected by the oxidative stress but their susceptibility may be different. This study aimed to investigate the antioxidative enzymes activated in EPCs after high constant glucose exposure as compared to endothelial cells (HUVECs). Both cells were incubated in the presence of normal (5mM) and high constant (25mM) d-glucose, as well as l-glucose as osmotic control for 48 and 96h. After a 48-hour exposure to high d-glucose, cell viability was significantly decreased both in EPCs and HUVECs as compared with normal d-glucose (p<0.01). However, after 96h there was no difference between EPCs grown on normal or high d-glucose, while HUVEC viability was affected by high d-glucose at 96h too (p<0.001). High d-glucose exposure induced a significant increase in reactive oxygen species (ROS) production in both cell types at 48h; however, after 96h, a significant decrease in ROS production (p<0.01) and a parallel marked increase in glutathione peroxidase type 1 (GPx-1) expression (p<0.01) and activity (p<0.01) were observed in EPCs compared to HUVECs. These data suggest that EPCs have a well-adaptive response to oxidative stress induced by constant and sustained high glucose exposure. This resistance to high glucose levels might be due to increased expression and activity of glutathione peroxidase allowing better cell survival.

Lack of association between endothelial nitric oxide synthase gene polymorphisms, microalbuminuria and endothelial dysfunction in hypertensive men

Background The Glu298Asp, T786C and 4a/4b genetic polymorphisms within the endothelial nitric oxide synthase (e-NOS) gene may predispose to hypertension, ischaemic heart disease and renal damage, possibly by reducing the generation of nitric oxide (NO), a fundamental substance in renal and cardiovascular biology. That same mechanism may contribute to raise albuminuria, a correlate of endothelial dysfunction and a marker of early kidney damage and poor cardiovascular prognosis in patients with hypertension. To assess that hypothesis, we evaluated the association of albuminuria with eNOS genotypes and their interacting potential with the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism. We also tested their impact on systemic NO availability, as reflected by endothelial-mediated forearm vasodilatation. Methods Albuminuria (three overnight collections), blood pressure, body mass index, renal function, glucose, lipids and prevalence of the metabolic syndrome were measured in 235 genetically unrelated, never-treated, uncomplicated white men with essential hypertension. Endothelial function was assessed in a patient subgroup (n = 94) by measuring plethysmographic forearm blood flow vasodilatation in response to intra-arterial acetylcholine with sodium nitroprusside as a control. Polymerase chain reaction or a 5′ nuclease assay were used to characterize the eNOS and ACE I/D variants. Results Albuminuria or microalbuminuria (albuminuria ≥ 15 μg/min) showed no association with eNOS polymorphisms either per se or after accounting for the co-existing ACE I/D genetic configuration. Forearm responses to acetylcholine did not differ by eNOS polymorphisms. Cardiovascular, renal, metabolic parameters were homogeneously distributed across different genetic backgrounds. Conclusion eNOS polymorphisms apparently play no role in promoting hypertensive renal damage, and do not influence endothelial-mediated vasodilatation in never-treated men with essential hypertension.

Grain and Bean Lysates Improve Function of Endothelial Progenitor Cells from Human Peripheral Blood: Involvement of the Endogenous Antioxidant Defenses

Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs), the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG), a Triticum Sativum grain powder, and Lady Joy (LJ), a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2). Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2)-like 2)/ARE (antioxidant response element) activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35–0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35–0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35–0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2) expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a protective effect of lysates on EPCs exposed to oxidative stress through the involvement of antioxidant systems. Lisosan G seems to activate the Nrf-2/ARE pathways.

Alpha-adducin and angiotensin-converting enzyme polymorphisms in hypertension: evidence for a joint influence on albuminuria.

Background A single-nucleotide polymorphism (Gly460Trp) within the α-adducin gene (ADD1) may influence several renal phenotypes, including salt sensitivity, susceptibility to renal failure, the renal haemodynamics and confer a worse cardiovascular risks profile. However, its relationship with microalbuminuria, a marker of early renal and cardiovascular damage and an independent predictor of morbid events in hypertension, is unknown. For this reason, we related the ADD1 genetic polymorphism to urine albumin levels and other clinical variables in essential hypertensive men. The angiotensin-converting enzyme (ACE) insertion/deletion (ID) polymorphism was also evaluated because of its interactive potential with the ADD1 genotype. Methods Albuminuria (three overnight collections), echocardiographic left ventricular mass index, blood pressure, body mass index, renal function, glucose and lipids were measured in 238 genetically unrelated, never treated, uncomplicated Caucasian essential hypertensive men. Polymerase chain reaction or a 5′ nuclease assay were used to characterize the ACE ID and ADD1 Gly460Trp variants, respectively. Results Microalbuminuria (albuminuria ≥ 15 μg/min) was more frequent in patients with the ACE DD variant, but only in those with a ADD1 Gly460Gly background. In contrast, urine albumin did not differ by ACE ID genotype in the presence of mutated ADD1 Trp alleles. ADD1 polymorphisms per se were not associated with albuminuria. Cardiovascular, renal, metabolic parameters were homogeneously distributed among different genetic backgrounds. Conclusions ACE DD and ADD1 Gly460Gly polymorphisms may jointly influence albuminuria in hypertensive men, 460Gly homozygosis facilitating or, possibly, the 460Trp allele mitigating the noxious renal impact of the ACE DD genotype. The data highlight further the complex pathophysiological implications of microalbuminuria in hypertension.

Cytochrome P450 2J2 Polymorphism in Healthy Caucasians and those with Diabetes Mellitus

AbstractObjective: Cytochrome P450 (CYP) 2J2 plays an important role in the biosynthesis of the biologically active cis-epoxyeicosatrienoic acids. An allelic variant named CYP2J2*6, which encodes an enzyme that is almost inactive in the metabolism of arachidonic acid, has recently been described. We investigated the frequency of the CYP2J2*6 variant in a Caucasian population and the relationship between this polymorphism and the development of micro- and macrovascular complications and hypertension in patients with type 1 or type 2 diabetes mellitus. Methods: Genomic DNA was extracted from peripheral blood cells and the fragment containing the A/T single nucleotide polymorphism at position 25 661 in exon 8 of the CYP2J2 gene was amplified. The 532 bp amplified product was subsequently digested with Tsp509I and analyzed on 12% polyacrylamide gel electrophoresis. Results: In the whole population, the frequency of the CYP2J2*6 allele was 0.0064 and the frequency of the CYP2J2*1 allele was 0.9936. Genotype distribution did not show significant differences between controls and patients with type 1 or type 2 diabetes. No homozygotes for CYP2J2*6 allele were found. No association was found between this allele and complications or hypertension in either type of diabetes. Conclusion: The CYP2J2*6 allele is rare in the Caucasian population, and no association is inferred between this allelic variant and diabetic complications.

IRS1 G972R Missense Polymorphism Is Associated With Failure to Oral Antidiabetes Drugs in White Patients With Type 2 Diabetes From Italy

This study tried to replicate in a large sample of white patients with type 2 diabetes (T2D) from Italy a previously reported association of the IRS1 G972R polymorphism with failure to oral antidiabetes drugs (OAD). A total of 2,409 patients from four independent studies were investigated. Case subjects (n = 1,193) were patients in whom, because of uncontrolled diabetes (i.e., HbA1c >8%), insulin therapy had been added either on, or instead of, maximal or near-maximal doses of OAD, mostly metformin and sulfonylureas; control subjects (n = 1,216) were patients with HbA1c <8% in the absence of insulin therapy. The IRS1 G972R polymorphism was typed by TaqMan allele discrimination. In all samples, individuals carrying the IRS1 R972 risk variant tended to be more frequent among case than control subjects, though reaching statistical significance only in one case. As no IRS1 G972R-by-study sample interaction was observed, data from the four samples were analyzed together; a significant association was observed (allelic odds ratio [OR] 1.30, 95% CI 1.03–1.63). When our present data were meta-analyzed with those obtained in a previous study, an overall R972 allelic OR of 1.37 (1.12–1.69) was observed. This study confirms in a large and ethnically homogeneous sample that IRS1 G972R polymorphism is associated with failure to OAD among patients with T2D.

Circulating endothelial progenitor cells in women with gestational alterations of glucose tolerance

Endothelial progenitor cells (EPCs) play a role in angiogenesis during pregnancy. The aim of this study was to evaluate circulating EPCs in pregnant women with gestational alterations of glucose tolerance. Glucose tolerance, insulin sensitivity and β-cell function were derived from oral glucose tolerance tests in 23 women with normal glucose tolerance (NGT), 18 with gestational impaired glucose tolerance (GIGT) and 24 with gestational diabetes mellitus (GDM). Circulating cells expressing CD34 in combination with CD133, kinase insert domain receptor (KDR) or both were quantified by flow cytometry. Women with GIGT and GDM had lower CD34+KDR+ and CD34+CD133 +KDR+ cells at 27±3.2 weeks’ gestation compared with NGT (ANOVA p<0.02 for both). CD34+KDR+ and CD34+CD133+KDR+ cells were inversely correlated with the area-under-the-glucose-curve (p<0.005, for both) and positively to insulin secretion-sensitivity index (p<0.05, for both). Alterations of glucose tolerance during pregnancy are associated with a decrease in EPCs. Hyperglycaemia might exert a direct effect on depletion of EPCs.

ACE GENE INSERTION/DELETION POLYMORPHISM MODULATES CAPILLARY PERMEABILITY IN HYPERTENSION

A D/D (deletion/deletion) polymorphism within the ACE (angiotensin 1-converting enzyme) gene increases the risk of microalbuminuria, a predictor of atherosclerotic vascular disease, in essential hypertension. It is unknown, however, whether this genetic profile is accompanied by disturbed macromolecular permeability of systemic capillary endothelium, possibly in the context of generalized endothelial dysfunction. In the present study, the ACE gene polymorphism was determined by PCR in 79 never-treated uncomplicated hypertensive men and 16 normotensive men as controls. Evaluation variables were TERalb (transcapillary escape rate of albumin; the 1-h decline rate of intravenous (125)I-albumin, a measure of integrity of systemic capillary endothelium), albuminuria and forearm vasodilation to intra-arterial acetylcholine, an index of NO (nitric oxide)-mediated vasomotion, in addition to a series of sensitive parameters of albumin permeation (blood pressure, metabolic status and smoking habits). Analyses were done by comparing D/D homozygotes with grouped I/D (insertion/deletion) and I/I (insertion/insertion) subjects. TERalb was higher in D/D hypertensives, who had higher albuminuria, more frequent microalbuminuria and comparable forearm responsiveness to intra-arterial acetylcholine. Fasting glucose and insulin, insulin sensitivity, 24-h blood pressure, smoking habits and metabolic parameters did not differ between the two groups. TERalb and urine albumin values were positively associated in the hypertensive subjects. In conclusion, ACE D/D homozygosis, independently of several confounding factors, associates with higher TERalb in men with essential hypertension. This may reflect noxious genetic influences on systemic vascular permeability, a critical control mechanism for atherogenesis in the absence of grossly impaired NO-mediated arteriolar responsiveness. The parallel behaviour of TERalb and albuminuria suggests some shared genetically mediated determinant of renal and systemic microvascular abnormalities in hypertension.

The 9p21 coronary artery disease locus and kidney dysfunction in patients with Type 2 diabetes mellitus

BACKGROUND We investigated whether the coronary artery disease (CAD) locus on chromosome 9p21 (as represented by single nucleotide polymorphism rs2383206) is associated with low estimated glomerular filtration rate (eGFR) or increased urinary albumin excretion in patients with Type 2 diabetes mellitus (T2DM). METHODS Four samples, including a total of 3167 patients, were studied. The presence of low eGFR (<60 mL/min/1.73m(2)) was estimated from serum creatinine by means of the Modification of Diet in Renal Disease Study equation. Increased urinary albumin excretion was defined as an albumin-creatinine ratio (ACR) ≥2.5 mg/mmol in men and ≥3.5 mg/mmol in women. RESULTS No association was found between rs2383206 and low eGFR or increased ACR in each sample as well as in a pooled analysis (overall odds ratio = 1.07, 95% confidence interval 0.94-1.22, P = 0.31 and overall odds ratio = 1.00, 95% confidence interval 0.90-1.12, P = 0.95, respectively). No interaction was observed between rs2383206 and poor glycemic control [HbA1c was above the median in the pooled sample (7.7%) in modulating eGFR or ACR (P for interaction = 0.42 and 0.90, respectively)]. CONCLUSION Variability at the 9p21 CAD locus is unlikely to play a role in modulating susceptibility to kidney dysfunction in patients with T2DM.

A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS) orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs), bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG), on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER) stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation.