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1.
Vascul Pharmacol ; 62(2): 49-56, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24699252

ABSTRACT

BACKGROUND: Thromboxane (TX) A2, prostaglandin endoperoxides and F2-isoprostanes exert their effects through a TX-prostanoid (TP) receptor, also expressed in endothelial cells. We investigated a role of the TP receptor in the endothelial expression of tissue factor (TF), a key trigger to thrombosis. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) exposed to the TP receptor agonist U46619 featured a concentration-dependent increase in TF surface exposure and procoagulant activity. HUVEC pre-incubation with the TP receptor antagonist S18886, followed by stimulation with either U46619 or tumor necrosis factor-α (TNF-α), attenuated TF surface exposure and activity compared with stimulated control. Aspirin or indomethacin, while inhibiting cyclooxygenase (COX)-1 and -2 activities, did not mimic this effect. Probing of underlying mechanisms by selective pharmacological and gene silencing experiments showed that S18886 reduced U46619- or TNF-α-induced TF expression inhibiting ROS production, NAD(P)H oxidase and PKC activation. In addition, S18886 also inhibited ERK activation in the presence of both U46619 and TNF-α alone, while inhibition of JNK activation only occurred in the presence of U46619. CONCLUSION: The endothelial TP receptor contributes to TF surface exposure and activity induced not only by known TP receptor agonists, but also by TNF-α. Such findings expand the therapeutic potential of TP receptor inhibition.


Subject(s)
Endothelium/metabolism , Receptors, Thromboxane/metabolism , Thromboplastin/metabolism , Tumor Necrosis Factor-alpha/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Cells, Cultured , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Endothelium/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , MAP Kinase Signaling System/drug effects , NADP/metabolism , Naphthalenes/pharmacology , Propionates/pharmacology , Protein Kinase C/metabolism , Reactive Oxygen Species/metabolism , Receptors, Thromboxane/agonists , Receptors, Thromboxane/antagonists & inhibitors , Signal Transduction/drug effects
2.
Curr Cardiol Rev ; 9(3): 253-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23909635

ABSTRACT

Biobanks play a crucial role in "-Omics" research providing well-annotated samples to study major diseases, their pathways and mechanisms. Accordingly, there are major efforts worldwide to professionalize biobanks in order to provide high quality preservation and storage of biological samples with potentially greater scientific impact. Biobanks are an important resource to elucidate relevant disease mechanisms as well as to improve the diagnosis, prognosis, and treatment of both pediatric and adult cardiovascular disease. High-quality biological sample collections housed in specialized bio-repositories are needed to discover new genetic factors and molecular mechanisms of congenital heart disease and inherited cardiomyopathies in order to prevent the potential risk of having a fatal cardiac condition as well as to facilitate rational drug design around molecular diseases (personalized medicine). Biological samples are also required to improve the understanding the environmental mechanisms of heart disease (environmental cardiology). The goal of this paper is to focus on preanalytical issues (informed consent, sample type, time of collection, temperature and processing procedure) related to collection of biological samples for research purposes. In addition, the paper provides an overview of the efforts made recently by our Institute in designing and implementing a high-security liquid nitrogen storage system (-196°C). We described the implementations of reliable preservation technologies and appropriate quality control (the right temperature, the right environment, fully traceable with all possible back-up systems) in order to ensure maximum security for personnel as well as the quality and suitability of the stored samples.


Subject(s)
Biological Specimen Banks , Cryopreservation/methods , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Biological Specimen Banks/economics , Humans , Metabolomics/methods , Nitrogen , Research Design , Specimen Handling/methods
3.
Recenti Prog Med ; 103(9): 307-12, 2012 Sep.
Article in Italian | MEDLINE | ID: mdl-23023017

ABSTRACT

Biobanks are a critical resource for "omics" technologies in order to dissect molecular mechanism and gene-environmental interactions of common diseases, such as cancer, cardiovascular diseases, diabetes, and neurodegenerative diseases. Progress in basic biomedicine may contribute to advance personalised medicine in which treatments will no longer be "one size fits all", but instead "tailored" to the molecular and genetic profile of each patient. Currently, there are major efforts worldwide to professionalize biobanks in order to move ahead from a "do-it-yourself" tissue collection - as is most frequent at present - for providing high quality preservation and storage of biological samples with potentially greater scientific impact. In this paper, we describe our recent experience in the design and development of a high-security liquid nitrogen storage system (-196°C) as a key resource for biomedical research.


Subject(s)
Biological Specimen Banks/organization & administration , Biomedical Research/organization & administration , Nitrogen , Plasma , Serum , Biological Specimen Banks/ethics , Biomedical Research/ethics , Blood , Humans , Italy , Metabolomics , Proteomics , Specimen Handling/standards , Transcriptome
4.
Nitric Oxide ; 21(3-4): 184-91, 2009.
Article in English | MEDLINE | ID: mdl-19654051

ABSTRACT

Decreased endothelial nitric oxide (NO) production and increased expression of vascular cell adhesion molecule-1 (VCAM-1) are early features of atherosclerosis. We investigated the effects of suppressing endogenous NO production by the NO synthase inhibitor l-mono-methyl-arginine (L-NMMA), given alone or in combination with interleukin(IL)-1alpha, on VCAM-1 expression by human umbilical vein endothelial cells (HUVEC). VCAM-1 expression (by enzyme immunoassay), barely detectable at baseline, was significantly increased by L-NMMA (by no more than 20% over control compared with IL-1alpha induction). This was paralleled by an increase in U937 monocytoid cell adhesion. When HUVEC incubated with L-NMMA were stimulated with low concentrations of IL-1alpha (0.05-0.5ng/mL), these determined a higher VCAM-1 expression than in the presence of L-NMMA or IL-1alpha alone. Northern analysis indicated that VCAM-1 mRNA was induced by L-NMMA alone, and that the effects of L-NMMA and IL-1alpha were, again, at least additive. Nuclear factor-kappaB (NF-kappaB), GATA, activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), transcription factors all involved in VCAM-1 gene expression, were all activated at electrophoretic mobility shift assay and at chromatin immunoprecipitation assay by L-NMMA, but additive effects with the combined administration of L-NMMA and IL-1alpha only occurred for NF-kappaB. These results support the view that endogenous NO mantains a normal endothelial non-reactivity towards circulating monocytes, and that suppression of this endogenous brake for endothelial activation results in the activation of multiple transcription factors even in the absence of other endothelial activators, with a prominent role of NF-kappaB in the presence or absence of other inflammatory mediators.


Subject(s)
Atherosclerosis/metabolism , Endothelium, Vascular/metabolism , NF-kappa B/metabolism , Nitric Oxide/physiology , Vascular Cell Adhesion Molecule-1/biosynthesis , Atherosclerosis/genetics , Cells, Cultured , Electrophoretic Mobility Shift Assay , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , GATA Transcription Factors/drug effects , GATA Transcription Factors/metabolism , Gene Expression Regulation , Humans , Interferon Regulatory Factor-1/drug effects , Interferon Regulatory Factor-1/metabolism , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/antagonists & inhibitors , Promoter Regions, Genetic , Transcription Factor AP-1/drug effects , Transcription Factor AP-1/metabolism , Vascular Cell Adhesion Molecule-1/genetics , omega-N-Methylarginine/pharmacology
5.
Coron Artery Dis ; 19(5): 299-305, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18607166

ABSTRACT

OBJECTIVE: In animal models, increased tissue receptor for advanced glycation end products and its ligands, including N-epsilon-(carboxymethyl)lysine (CML), are critically implicated in postprocedural intimal hyperplasia after balloon injury. In patients undergoing percutaneous coronary interventions with stenting, we investigated whether plasma levels of CML and the soluble form of receptor for advanced glycation end products (sRAGE) changed during poststenting follow-up. METHODS: We studied 81 patients with coronary artery disease who underwent successful percutaneous coronary interventions. Plasma levels of CML and sRAGE were measured before intervention, and at 1 day and 180 days of follow-up. RESULTS: CML levels increased significantly at day 1 after stenting and persisted at an elevated level at 180 days (P=0.013), whereas sRAGE levels increased significantly at 180 days (P=0.011). CML levels were significantly higher in multivessel-treated patients than in single-vessel-treated patients both at 1 day and 180 days of follow-up. In addition, CML values were positively associated with the extent of stent area at 1 day and 180 days of follow-up (r=0.278, P=0.022 and r=0.315, P=0.012, respectively). In logistic regression analysis, only the extent of stent area predicted adverse clinical events at 180-day follow-up (P=0.03, odds ratio=14.25, confidence interval=1.25-162.2). CONCLUSION: This study supports the hypothesis that increased circulating levels of CML occurred in the presence of vascular injury. This persistent rise of CML could amplify an inflammatory phenomenon triggered by stent placement and thus contributes to coronary artery disease progression.


Subject(s)
Angina, Unstable/therapy , Angioplasty, Balloon, Coronary , Lysine/analogs & derivatives , Receptors, Immunologic/blood , Stents/adverse effects , Aged , C-Reactive Protein , Catheterization/adverse effects , Female , Follow-Up Studies , Humans , Logistic Models , Lysine/blood , Male , Middle Aged , Predictive Value of Tests , Receptor for Advanced Glycation End Products , Treatment Outcome
6.
Haematologica ; 93(6): 892-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18469350

ABSTRACT

BACKGROUND: Increased levels of microparticles exposing tissue factor circulate in the blood of patients with coronary heart disease, possibly disseminating their pro-thrombotic and pro-inflammatory potential. Because diets rich in n-3 (polyunsaturated) fatty acids have been associated with reduced incidence of coronary heart disease-related events, we investigated the in vivo effects of treatments with n-3 fatty acids on levels of circulating microparticles and their tissue factor- dependent procoagulant activity in patients with a previous myocardial infarction. DESIGN AND METHODS: Forty-six post-myocardial infarction patients were assigned to receive either 5.2 g of n-3 fatty acids daily (n=23) or an olive oil placebo (n = 23) for 12 weeks. Circulating microparticles were isolated from peripheral blood. The number of microparticles, their cellular source and tissue factor antigen were determined by flow cytometry, and their procoagulant potential assayed by a fibrin generation test. RESULTS: The total number of microparticles, endothelium-derived microparticles and microparticle tissue factor antigen were not significantly different between the two groups. However, the number of platelet-derived microparticles [from a median of 431 (126-1796, range) x 10(6)/L to a median of 226 (87-677, range)] x 10(6)/L and monocyte-derived microparticles [from a median of 388 (9-1681, range) x 10(6)/L to a median of 265 (7-984, range) x 10(6)/L] in plasma were significantly (p < 0.05) decreased by n-3 fatty acids, while they were unchanged in the placebo group. Total microparticle tissue factor-procoagulant activity was also reduced in the n-3 fatty acid group compared to that in the placebo group. CONCLUSIONS: Treatment with n-3 fatty acids after myocardial infarction exerts favorable effects on levels of platelet- and monocyte-derived microparticles, thus possibly explaining some of the anti-inflammatory and anti-thrombotic properties of these natural compounds.


Subject(s)
Fatty Acids, Omega-3/chemistry , Fatty Acids, Unsaturated/metabolism , Myocardial Infarction/blood , Myocardial Infarction/metabolism , Aged , Blood Platelets/metabolism , Coronary Disease/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Female , Fibrin/chemistry , Humans , Male , Middle Aged , Placebos , Thromboplastin/metabolism , Thrombosis/metabolism
7.
Thromb Haemost ; 98(1): 210-9, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17598015

ABSTRACT

Tissue factor (TF) is expressed on the endothelium in response to inflammatory mediators, giving endothelial cells a pro-thrombotic phenotype. Since fish-derived n-3 fatty acids (FA) have been associated with reduced incidence of myocardial infarction, we investigated the endothelial effects of the most abundant n-3 FA, docosahexaenoate (DHA), on TF expression. Human umbilical vein endothelial cells were pre-incubated with DHA (or stearate and arachidonate as controls) for 48-72 hours, and then stimulated with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Pre-incubation of endothelial cells with DHA (but not stearate or arachidonate) concentration-dependently reduced surface protein exposure, independent of TF mRNA or total protein expression regulation. Conversely, DHA treatment in conjunction with activating stimuli, induced the release of endothelial TF-exposing microparticles from endothelial cells, quantitatively accounting for the decreased TF cell surface exposure. In conclusion, DHA treatment, with a time-course consistent with its incorporation in membrane phospholipids, increases the release of TF-exposing microparticles from endothelial cells, accounting for decreased endothelial cell TF surface exposure, thus potentially modifying the overall endothelial control of microparticle-related effects.


Subject(s)
Docosahexaenoic Acids/pharmacology , Endothelial Cells/physiology , Thromboplastin/metabolism , Cells, Cultured , Endothelium, Vascular/cytology , Fatty Acids, Omega-3/pharmacology , Humans , Lipopolysaccharides , Membrane Proteins/metabolism , Tumor Necrosis Factor-alpha , Umbilical Veins/cytology
8.
Eur Heart J ; 28(4): 484-91, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17267459

ABSTRACT

AIMS: The contribution of homocysteine and group B vitamins in determining cardiovascular risk is debated. We assessed the predictive value of total homocysteine (tHcy), vitamin B12, folate, and vitamin B6 on the long-term occurrence of coronary and cerebral atherothrombotic events in a nested case-control study. METHODS AND RESULTS: Within a cohort of 1021 healthy subjects (490 men and 531 women) recruited in 1987, 66 first-ever coronary and 43 first-ever cerebrovascular events were recorded at a 12-year follow-up (cases, n=109). A total of 109 control subjects (remaining free from events) were matched with cases according to age, sex, smoking, hypertension, dyslipidaemia, and body mass index. Serum samples obtained in 1987 at baseline were used to measure tHcy, folate, and vitamins B12 and B6, as well as C-reactive protein plasma concentrations. We found a significant graded association between tHcy levels and the risk of coronary and cerebrovascular events [odds ratio (OR) for uppermost vs. lowermost quartile=1.34, 95% CI 1.01-1.76)]. Folate and vitamin B12 did not significantly differ between cases and controls, but were negatively (P<0.01) correlated with tHcy. Vitamin B6 did not correlate with tHcy levels, but differed significantly between cases and controls: for subjects in the uppermost quartile vs. the lowermost quartile of vitamin B6, OR=0.69 (95% CI 0.49-0.98). For subjects in the lowermost quartile of vitamin B6 and the uppermost quartile of tHcy, OR=17.50 (95% CI 1.97, 155.59). Cases and controls were not different as to C-reactive protein. CONCLUSION: tHcy and plasma vitamin B6 are long-term independent risk factors for coronary and cerebrovascular events.


Subject(s)
Coronary Artery Disease/blood , Coronary Thrombosis/blood , Homocysteine/metabolism , Intracranial Arteriosclerosis/blood , Intracranial Thrombosis/blood , Vitamin B 6/metabolism , Adult , C-Reactive Protein/metabolism , Case-Control Studies , Coronary Artery Disease/etiology , Coronary Thrombosis/etiology , Female , Humans , Intracranial Arteriosclerosis/etiology , Intracranial Thrombosis/etiology , Male , Middle Aged , Prospective Studies , Risk Factors , Stroke/blood , Stroke/etiology
9.
Proc Natl Acad Sci U S A ; 103(41): 15184-9, 2006 Oct 10.
Article in English | MEDLINE | ID: mdl-17018645

ABSTRACT

A high intake of the omega-3 fatty acid docosahexaenoate [docosahexaenoic acid (DHA)] has been associated with systemic antiinflammatory effects and cardiovascular protection. Cyclooxygenase (COX)-2 is responsible for the overproduction of prostaglandins (PG) at inflammatory sites, and its expression is increased in atheroma. We studied the effects of DHA on COX-2 expression and activity in human saphenous vein endothelial cells challenged with proinflammatory stimuli. A>or=24-h exposure to DHA reduced COX-2 expression and activity induced by IL-1, without affecting COX-1 expression. DHA effect depended on the NF-kappaB-binding site in the COX-2 promoter. EMSAs confirmed that DHA attenuated NF-kappaB activation. Because MAPK, PKC, and NAD(P)H oxidase all participate in IL-1-mediated COX-2 expression, we also tested whether these enzymes were involved in DHA effects. Western blots showed that DHA blocked nuclear p65 NF-kappaB subunit translocation by decreasing cytokine-stimulated reactive oxygen species and ERK1/2 activation by effects on both NAD(P)H oxidase and PKCepsilon activities. Finally, to address the question whether DHA itself or DHA-derived products were responsible for these effects, we inhibited the most important enzymes involved in polyunsaturated fatty acid metabolism, showing that 15-lipoxygenase-1 products mediate part of DHA effects. These studies provide a mechanistic basis for antiinflammatory and possibly plaque-stabilizing effects of DHA.


Subject(s)
Cyclooxygenase 2/metabolism , Docosahexaenoic Acids/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , NADPH Oxidases/antagonists & inhibitors , Protein Kinase C-epsilon/antagonists & inhibitors , Cells, Cultured , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2 Inhibitors/pharmacology , Enzyme Induction/drug effects , Enzyme Induction/physiology , Humans
10.
J Clin Endocrinol Metab ; 91(11): 4628-34, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16926247

ABSTRACT

CONTEXT: The interaction of advanced glycation end products, including Nepsilon-(carboxymethyl)lysine-protein adducts (CML) and S100A12 protein, with their cellular receptor (RAGE) is implicated in the pathogenesis of diabetic vascular complications. RAGE has a circulating secretory receptor form, soluble RAGE (sRAGE), which, by neutralizing the action of advanced glycation end products, might exert a protective role against the development of cardiovascular disease. OBJECTIVE: The objective of the study was to investigate whether plasma sRAGE levels are associated with glycemic control, proinflammatory factors, or circulating ligands of RAGE such as plasma CML and S100A12 protein. STUDY DESIGN: We studied 160 subjects, 84 subjects with type 2 diabetes (aged 60 +/- 7 yr) and 76 nondiabetic controls (aged 45 +/- 10 yr). RESULTS: Plasma sRAGE was lower in diabetic patients than controls [141 (53-345) vs. 735 (519-1001) pg/ml, median (interquartile range), P < 0.0001], whereas CML levels were higher in diabetic patients than controls [67.9 (46.0-84.7) vs. 43.4 (28.0-65.0) microg/ml, P < 0.0001]. In stepwise regression analysis of the whole data set, hemoglobin A1c, insulin resistance (as homeostasis model assessment), and C-reactive protein were independently associated with plasma sRAGE, whereas age was not. In a subgroup of 26 diabetic and 24 nondiabetic subjects of similar age (54 +/- 3 yr), plasma S100A12 levels were higher in diabetic subjects [49 (39-126) vs. 28 (21-39) ng/ml]. Moreover, low sRAGE and high S100A12 were strongly associated with increased risk for cardiovascular disease (Framingham score). In this subgroup, the plasma S100A12 level was the only determinant of plasma sRAGE concentration. CONCLUSION: Plasma level of sRAGE is down-regulated in chronic hyperglycemia; among its ligands, S100A12 protein, but not CML, appears to be associated with this effect.


Subject(s)
Blood Glucose/analysis , Receptors, Immunologic/blood , S100 Proteins/blood , Adult , Aged , Blood Pressure , C-Reactive Protein/analysis , Case-Control Studies , Diabetes Mellitus, Type 2/blood , Diabetic Angiopathies/blood , Diabetic Angiopathies/epidemiology , Female , Glycated Hemoglobin/analysis , Glycation End Products, Advanced/blood , Glycation End Products, Advanced/metabolism , Humans , Inflammation Mediators/blood , Lysine/analogs & derivatives , Lysine/blood , Male , Middle Aged , Receptor for Advanced Glycation End Products , Risk Factors , S100A12 Protein , Statistics as Topic
11.
Microvasc Res ; 72(1-2): 12-9, 2006.
Article in English | MEDLINE | ID: mdl-16750838

ABSTRACT

Oxidative stress and angiogenesis are important elements in the pathogenesis of atherosclerosis and cancer. Because of its antioxidant properties, alpha-tocopherol has long proposed as prevention of diseases associated with oxidative stress. We explore whether alpha-tocopherol modulates some cell responses induced by angiogenic and proliferative stimuli. For this purpose, we evaluate the effect in human vein endothelial cells (HUVECs), of alpha-tocopherol treatment (5-40 micromol/L) for 72 h on the production of reactive oxygen species (ROS), induction of matrix metalloproteinases (MMPs), expression of vascular endothelial-cadherin (VE-cadherin) and alpha(2)-integrin, cell migration, cell proliferation, and tube formation. alpha-Tocopherol significantly inhibits intracellular ROS production induced by TNF-alpha (P < 0.01) or PMA (P < 0.001). However, alpha-tocopherol does not interfere with mRNA expression of VE-cadherin, alpha(2)-integrin, MMP-1, MMP-2, and MMP-9. Similarly, alpha-tocopherol does not modulate cell migration and capillary-like tube formation although at the concentration of 20 and 40 micromol/L it potentiated PMA-induced DNA synthesis (P < 0.05). Our results suggest that although alpha-tocopherol supplementation reduces endothelial cell oxidative stress, it does not alter the cell response to angiogenic stimuli.


Subject(s)
Neovascularization, Pathologic , alpha-Tocopherol/pharmacology , Antigens, CD/metabolism , Bromodeoxyuridine/pharmacology , Cadherins/metabolism , Cell Movement , Cell Proliferation , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Integrins/metabolism , Matrix Metalloproteinases/metabolism , Microcirculation , Oxidative Stress , Reactive Oxygen Species
12.
Am J Physiol Heart Circ Physiol ; 291(4): H1814-21, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16648189

ABSTRACT

A paradoxical microcirculatory constriction has been observed in hearts of patients with ischemia, secondary to coronary stenosis. Here, using the isolated mouse heart (Langendorff), we examined the mechanism of this response, assuming involvement of nitric oxide (NO) and endothelin-1 (ET-1) systems. Perfusion pressure was maintained at 65 mmHg for 70 min (protocol 1), or it was reduced to 30 mmHg over two intervals, between the 20- and 40-min marks (protocol 2) or from the 20-min mark onward (protocol 3). In protocol 1, coronary resistance (CR) remained steady in untreated heart, whereas it progressively increased during treatment with the NO synthesis inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) (2.7-fold) or the ET(A) antagonist BQ-610 (2.8 fold). The ET(B) antagonist BQ-788 had instead no effect by itself but curtailed vasoconstriction to BQ-610. In protocol 2, hypotension raised CR by 2.2-fold. This response was blunted by reactive oxygen species (ROS) scavengers (mannitol and superoxide dismutase plus catalase) and was converted into vasodilation by l-NAME, BQ-610, or BQ-788. Restoration of normal pressure was followed by vasodilation and vasoconstriction, respectively, in untreated and treated preparations. In protocol 3, CR progressively increased with hypotension in the absence but not presence of L-NAME or BQ-610. We conclude that the coronary vasculature is normally relaxed by two concerted processes, a direct action of NO and ET-1 curtailing an ET(B2)-mediated tonic vasoconstriction through ET(A) activation. The negative feedback mechanism on ET(B2) subsides during hypotension, and the ensuing vasoconstriction is ascribed to ET-1 activating ET(A) and ET(B2) and reactive nitrogen oxide species originating from ROS-NO interaction.


Subject(s)
Coronary Stenosis/physiopathology , Coronary Vessels/physiopathology , Endothelins/physiology , Myocardial Ischemia/physiopathology , Nitric Oxide/physiology , Animals , Antihypertensive Agents/pharmacology , Blood Pressure/physiology , Coronary Stenosis/complications , Enzyme Inhibitors/pharmacology , Hemodynamics/physiology , Hypotension/physiopathology , Mice , Mice, Inbred C57BL , Microcirculation/physiopathology , Models, Cardiovascular , Myocardial Ischemia/etiology , NG-Nitroarginine Methyl Ester/pharmacology , Oligopeptides/pharmacology , Piperidines/pharmacology , Reactive Oxygen Species/metabolism , Vasoconstriction/drug effects , Vasoconstriction/physiology , Vasodilation/drug effects , Vasodilation/physiology , Vasomotor System/physiology
13.
Anal Bioanal Chem ; 384(3): 751-60, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16372182

ABSTRACT

Boron neutron capture therapy (BNCT) is a bimodal radiotherapeutic treatment based on the irradiation of neoplastic tissues with neutrons after the tissues have selectively accumulated molecules loaded with nuclides with large neutron capture cross-sections (such boron-10). Boron-10 carriers have been tested to a limited extent, and clinical trials have been conducted on sulfhydryl borane (10B-BSH) and boronophenylalanine (10B-BPA). However, precise and accurate measurements of boron-10 concentrations (0.1-100 microg/g) in specimens and samples of limited size (microg scale) are needed in order to be able to biologically characterise new compounds in predictive tissue dosimetry, toxicology and pharmacology studies as well as in clinical investigations. A new approach based on fast separation and detection of 10B-BPA performed by coupling capillary electrophoresis to electrospray mass spectrometry is reported. This method allows the quantitative analysis and characterisation of 10B-BPA in a short time with a high separation efficiency. Detection limits of 3 microM for 10B-BPA and 30 ng/mL for 10B were obtained with CE-ESI-MS. A quantification limit of 10 microM for 10B-BPA (100 ng/mL for 10B) was attained. The total boron-10 concentration was determined by high-resolution inductively coupled mass spectrometry in order to validate the method. Boron-10 isotope measurements were carried out by HR-ICP-MS at medium resolution (R=4000) due to the presence of an isobaric interference at mass 10. Good agreement was obtained between the values from CE-ESI-MS and those from HR-ICP-MS. The method has been successfully used to determine the 10B-BPA in two lines of cultured cells.


Subject(s)
Boron Compounds/analysis , Boron Neutron Capture Therapy , Endothelial Cells/chemistry , Glioma/chemistry , Mass Spectrometry/methods , Phenylalanine/analogs & derivatives , Animals , Boron Compounds/pharmacokinetics , Cell Line, Tumor , Electrophoresis, Capillary , Endothelial Cells/metabolism , Glioma/metabolism , Humans , Isotopes , Molecular Structure , Phenylalanine/analysis , Phenylalanine/pharmacokinetics , Rats , Sensitivity and Specificity , Time Factors
14.
Arterioscler Thromb Vasc Biol ; 25(7): 1401-7, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15845907

ABSTRACT

OBJECTIVE: The interaction of advanced glycation end products (AGEs) with their main receptor RAGE in endothelial cells induces intracellular generation of reactive oxygen species (ROS) and the expression of vascular cell adhesion molecule (VCAM)-1. We investigated the role of distinct sources of ROS, including the mitochondrial electron transport chain, NAD(P)H oxidase, xanthine oxidase, and arachidonic acid metabolism, in AGE-induced VCAM-1 expression. METHODS AND RESULTS: The induction of ROS and VCAM-1 by AGEs in cultured human umbilical vein endothelial cells was specifically blocked by an anti-RAGE antibody. The inhibition of NAD(P)H oxidase by apocynin and diphenylene iodonium, and of the mitochondrial electron transport system at complex II by thenoyltrifluoroacetone (TTFA), significantly inhibited both AGE-induced ROS production and VCAM-1 expression, whereas these effects were potentiated by rotenone and antimycin A, specific inhibitors of mitochondrial complex I and III, respectively. The inhibition of Cu/Zn superoxide dismutase inhibited both ROS and VCAM-1 induction, indicating that H2O2 by this source is involved as a mediator of VCAM-1 expression by AGEs. CONCLUSIONS: Altogether, these results demonstrate that ROS generated by both NAD(P)H-oxidase and the mitochondrial electron transport system are involved in AGE signaling through RAGE, and indicate potential targets for the inhibition of the atherogenic signals triggered by AGE-RAGE interaction.


Subject(s)
Atherosclerosis/metabolism , Endothelium, Vascular/metabolism , Glycation End Products, Advanced/metabolism , Reactive Oxygen Species/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Arachidonic Acid/metabolism , Arachidonic Acid/pharmacology , Cell Nucleus/metabolism , Cells, Cultured , Cytoplasm/enzymology , Electron Transport/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Glycation End Products, Advanced/pharmacology , Humans , Mitochondria/enzymology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Receptor for Advanced Glycation End Products , Receptors, Immunologic/metabolism , Signal Transduction/physiology , Superoxide Dismutase/antagonists & inhibitors , Superoxide Dismutase/metabolism , Transcription Factor RelA/metabolism , Umbilical Veins/cytology , Xanthine Oxidase/antagonists & inhibitors , Xanthine Oxidase/metabolism
15.
J Cardiovasc Pharmacol ; 44(3): 356-62, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15475834

ABSTRACT

The effect of Ginkgo biloba extract (EGb 761) was studied in rat hearts submitted to ischemia/reperfusion. Isolated hearts perfused in Langendorff mode were subjected to 60 minutes of global ischemia and 15 minutes of reperfusion. EGb 761 was administered by chronic or acute treatment: intra-peritoneal injections of 5 mg/Kg extract for 5 days, or 100 mg /L extract addition to the perfusion buffer, respectively. In hearts not treated with EGb 761, ischemia induced a 20% decrease in the concentration of membrane alpha-tocopherol. This effect was not worsened by reperfusion. alpha-tocopherol consumption was accompanied by about 650% increase in 6-ketoPGF1alpha release within 3 minutes of reperfusion. Moreover, ischemia induced activation of transcription factor NF-kappaB, as compared with the untreated group. In both chronic and acute treatment with EGb 761, heart concentration of alpha-tocopherol was completely spared during ischemia as much as after reperfusion, and a significant decrease of 6-ketoPGF1alpha release was observed at 3 minutes of reperfusion. Nuclear translocation of NF-kappaB was lowered during ischemia. EGb 761 might act as direct free radical scavenger or as tocopheryl radical recycler; in both cases sparing membrane vitamin E should affect phospholipase A2 activity. Finally, EGb 761, by lowering ROS produced during ischemia, challenges nuclear translocation of NF-kappaB.


Subject(s)
6-Ketoprostaglandin F1 alpha/biosynthesis , Myocardial Ischemia/drug therapy , Myocardial Reperfusion Injury/drug therapy , Plant Extracts/pharmacology , Vitamin E/metabolism , 6-Ketoprostaglandin F1 alpha/chemistry , 6-Ketoprostaglandin F1 alpha/metabolism , Animals , Cell Nucleus/metabolism , Electrophoretic Mobility Shift Assay/methods , Female , Freeze Drying , Ginkgo biloba , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , Male , Myocardial Ischemia/metabolism , Myocardial Ischemia/physiopathology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/physiopathology , Myocardium/chemistry , Myocardium/cytology , Myocardium/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Oxidative Stress/drug effects , Perfusion , Plant Extracts/chemistry , Plant Extracts/therapeutic use , Rats , Rats, Wistar , Transcription Factors/chemistry , Transcription Factors/metabolism , Vitamin E/chemistry
16.
Thromb Haemost ; 91(6): 1078-83, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15175792

ABSTRACT

If delivered at elevated intensity, ultrasound potentiates enzymatic clot dissolution; however, an elevated acoustic intensity damages vascular wall and favors reocclusion. This study's aim was to investigate whether exposure to high-frequency, low-intensity ultrasound - generated by a diagnostic scanner -enhances enzymatic thrombolysis, and if this effect differs in clots from blood of normal subjects and of patients with coronary artery disease (CAD). Venous blood samples were drawn from 10 healthy volunteers and from 10 CAD patients on chronic medical treatment, which also included aspirin. Each sample generated 2 radiolabelled clots, which were positioned in 2 in vitro models filled with human plasma recirculating at 37 degrees. One clot was exposed to acetyl salicylic acid (60 microg/ml), tissue plasminogen activator (3 microg/ml) and heparin (1 IU/ml), while the other was exposed to the same medications plus ultra-sound (2.5 MHz, mechanical index = 1.0) for 3 hours. Enzymatic thrombolysis was measured as solubilization of radiolabel. Normal subjects and patients did not significantly differ as to coagulation parameters, weight, volume and density of the clots, and fibrinolytic activity (p = 0.794). Ultrasound exposure did not influence thrombolysis in clots of normal subjects (p = 0.367), while it enhanced the dissolution of clots of CAD patients (p = 0.013). The enhancement was equal to 51% at 5 minutes, 32% at 15 minutes, 27% at 30 minutes, 20% at 1 hour and 19% at 3 hours (p < 0.05). Diagnostic ultrasound enhances enzymatic dissolution of clots generated from the blood of CAD patients, likely due to chronic treatment and in particular to aspirin.


Subject(s)
Thrombolytic Therapy/methods , Thrombosis/diagnostic imaging , Thrombosis/therapy , Ultrasonic Therapy/methods , Adult , Aged , Aged, 80 and over , Cardiovascular Diseases/blood , Case-Control Studies , Combined Modality Therapy , Drug Therapy, Combination , Female , Fibrinolytic Agents/therapeutic use , Humans , Kinetics , Male , Middle Aged , Models, Biological , Ultrasonic Therapy/instrumentation , Ultrasonography
17.
Am Heart J ; 148(1): e6, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15215815

ABSTRACT

BACKGROUND: We evaluated the short-term safety and efficacy of aspirin-plus-clopidogrel as antithrombotic therapy in nonvalvular atrial fibrillation (AF). METHODS AND RESULTS: Thirty patients (11 women, 45 to 75 years of age) with non-high-risk permanent (n = 12) or persistent AF awaiting cardioversion (n = 18) underwent transesophageal echocardiography to exclude left heart thrombi and were then randomly assigned to receive warfarin (international normalized ratio, 2 to 3 for 3 weeks) or aspirin (100 mg/d alone for 1 week)-plus-clopidogrel (75 mg/d added to aspirin for 3 weeks). Bleeding time and serum thromboxane B2 were measured at entry and at 3 weeks. Bleeding time, not affected by warfarin, was prolonged by 71% by aspirin (P <.05) and further, by 144%, by adding clopidogrel (P <.01 vs aspirin alone; +319%, P <.01, vs baseline). Thromboxane B2, not affected by warfarin, was reduced by aspirin (-98%, P <.01) but not further by clopidogrel. No thrombi or dense spontaneous echo-contrast were found at the 3-week transesophageal echocardiography. Seven of 9 patients receiving warfarin and 7 of 9 patients receiving aspirin-plus-clopidogrel, undergoing electrical cardioversion, achieved sinus rhythm. No thromboembolic or hemorrhagic events occurred in both arms throughout the 3-week treatment and a further 3-month follow-up. CONCLUSIONS: Aspirin-plus-clopidogrel and warfarin were equally safe and effective in preventing thromboembolism in this small group of patients with non-high-risk AF.


Subject(s)
Aspirin/therapeutic use , Atrial Fibrillation/drug therapy , Platelet Aggregation Inhibitors/therapeutic use , Thromboembolism/prevention & control , Ticlopidine/analogs & derivatives , Ticlopidine/therapeutic use , Aged , Anticoagulants/therapeutic use , Atrial Fibrillation/complications , Atrial Fibrillation/therapy , Bleeding Time , Clopidogrel , Drug Therapy, Combination , Echocardiography, Transesophageal , Electric Countershock , Female , Humans , International Normalized Ratio , Male , Middle Aged , Pilot Projects , Thromboxane B2/blood , Warfarin/therapeutic use
18.
Ital Heart J Suppl ; 4(5): 407-14, 2003 May.
Article in Italian | MEDLINE | ID: mdl-12848078

ABSTRACT

Therapeutic applications of ultrasound are currently limited to dental plaque removal, physiotherapy and lithotripsy. However, several in vitro and experimental studies have shown the ability of ultrasound to accelerate clot dissolution. This effect is mainly influenced by the intensity and frequency of the beam. High ultrasound energies, although effective, can induce early reocclusion, while moderate intensities and low ultrasound frequencies are better tolerated and equally effective. So far, few patients with acute myocardial infarction have been treated by ultrasound catheters. In patients with ischemic stroke, transcranial Doppler was monitored during the venous administration of recombinant tissue-type plasminogen activator (rt-PA) and the occluded vessel was recanalyzed earlier than in other studies by rt-PA alone (without Doppler monitoring). Ultrasound-accelerated thrombolysis is caused by a strengthening of the enzymatic action, favored by acoustic cavitation. As the microbubbles of echocontrast agents lower the cavitation threshold, they can further enhance the thrombolytic process. New generation microbubbles, able to bind to the thrombus surface, could facilitate thrombus-microbubbles interaction. The combination of ultrasound, microbubbles and fibrinolytic agents could benefit the treatment of a variety of cardiovascular diseases.


Subject(s)
Thrombolytic Therapy/methods , Gases , Humans , Time Factors , Ultrasonography
19.
Cardiovasc Res ; 58(1): 156-61, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12667957

ABSTRACT

Diagnostic cardiac ultrasound are commonly assumed to pose no hazard to the patient-but this is not synonymous with being biologically inert. The production of intracellular reactive oxygen species (ROS) on endothelial cells is a key modulator of atheroprotective (at low level) and atherogenic (at high levels) actions. The aim of the study was to evaluate in vitro the effects on intracellular ROS of endothelial cells after ultrasound exposure of variable duration with commercially available cardiac imaging systems. Endothelial cells fluorescence was evaluated in vitro after sham (transducer off) exposure to ultrasound and after 5', 15' and 30' of ultrasound irradiation with second harmonic 1.3/2.6 MHz cardiac ultrasound scan (mechanical index 1.5). Intracellular ROS were 83 at baseline, and rose to 86, 112 and 122 fluorescence intensity at 1 h incubation after 5', 15' and 30' of ultrasound exposure respectively (P<0.01 for 30' versus baseline and 5' comparison). There were microscopic signs of endothelial damage only following 30' stage. Ultrasound exposure induced significant DNA laddering and LDH leakage after 15' of ultrasound exposure. Effects on endothelial cells could be reproduced by adding exposed extracellular medium to unexposed cells, and could be prevented removing exposed medium from cell culture or pretreating the medium with catalase. Cardiac ultrasound of current clinical diagnostic use increases intracellular oxidative stress on endothelial cells in vitro. This increase is accompanied by morphological evidence of endothelial damage only after longer exposure times, persists 1 h after withdrawal of ultrasound, and can be modulated over a wide range according to the duration of ultrasound exposure. Free radical production in the extracellular medium is the likely mediator of ultrasound effect.


Subject(s)
Echocardiography/adverse effects , Endothelium, Vascular/metabolism , Oxidative Stress , Reactive Oxygen Species/metabolism , Apoptosis , Cell Membrane/diagnostic imaging , Cells, Cultured , DNA Fragmentation , Endothelium, Vascular/pathology , Free Radicals/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Time Factors
20.
Circulation ; 106(20): 2543-9, 2002 Nov 12.
Article in English | MEDLINE | ID: mdl-12427649

ABSTRACT

BACKGROUND: Both statins and vitamin E, by reducing the rate of lipid peroxidation, may interfere with oxidative stress, but the impact of their combination is unknown. METHODS AND RESULTS: We randomized 43 hypercholesterolemic patients (21 men, 22 women, age 63+/-11 years) to either simvastatin, to achieve >20% reduction of total cholesterol, or simvastatin plus 600 mg/d vitamin E for 2 months. Patients were then crossed over to the alternative treatment. Lipid parameters documented patients' compliance to simvastatin, whereas plasma levels of vitamin E documented compliance and absorption of vitamin E. We assessed urinary excretion of the isoprostane 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) as an in vivo index of oxidative stress at baseline and after each month of therapy. 8-Iso-PGF(2alpha) was significantly reduced by simvastatin, from 361+/-148 pg/mg creatinine (mean+/-SD) at baseline to 239+/-124 pg/mg creatinine after 1 month. The addition of vitamin E did not reduce such levels any further (256+/-125 after 1 month). Linear regression analysis showed a weak inverse relationship of 8-iso-PGF(2alpha) with vitamin E levels but a much stronger relationship with LDL cholesterol (R(2)=0.162; P<0.001). CONCLUSIONS: In hypercholesterolemic patients, LDL cholesterol is a major correlate of oxidative stress. Concomitant with LDL cholesterol reduction, simvastatin causes a drastic reduction of oxidative stress to a level that is not further reduced by the addition of vitamin E. Results of clinical trials with vitamin E may have been hampered by inadequate knowledge of the background level of lipid peroxidation, which is a major determinant of vitamin E bioactivity.


Subject(s)
Cholesterol, LDL/blood , Dinoprost/analogs & derivatives , F2-Isoprostanes/urine , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypercholesterolemia/drug therapy , Simvastatin/therapeutic use , Vitamin E/therapeutic use , Antioxidants/therapeutic use , Cholesterol/blood , Cross-Over Studies , Drug Therapy, Combination , Female , Humans , Hypercholesterolemia/blood , Hypercholesterolemia/urine , Lipid Peroxidation/drug effects , Lipoproteins, LDL/blood , Male , Middle Aged , Oxidative Stress , Patient Compliance , Vitamin E/blood
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