Luigi Iuliano

Localization of distinct F2-isoprostanes in human atherosclerotic lesions.

F2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

Oxygen free radicals and platelet activation

This article reviews our current understanding of the role of oxygen free radicals in platelet activation. Several studies have indicated that platelets, in analogy to other circulating blood cells, are able to produce oxygen free radicals, which are likely to play an important role in the mechanism of platelet activation and aggregation. Platelet activation has been obtained with very low, physiologically relevant concentrations of radicals generated chemically, by leukocytes, and by hemoglobin derived from membrane leakage of erythrocytes. Knowledge of the role of reactive species in platelet physiology is relevant because platelets are brought into close contact with other cells capable of producing free radicals, such as neutrophils, macrophages, and endothelial cells, during the formation of thrombus. The physiopatological importance of these findings is high because it is now emerging that free radicals may have a role in the mechanism of atherosclerosis and its thrombotic complications, where the causative role of platelets is well documented. This background suggests therapeutic interventions with antioxidants as antiplatelet agents to improve the pharmacological effect of classical antiplatelet drug such as aspirin.

Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication

In patients with intermittent claudication, exercise is associated with a marked increase in oxidative stress, likely responsible for systemic endothelial perturbation. In 31 claudicant patients, we assessed the effect of vitamin C administration on the acute changes induced by maximal and submaximal exercise in endothelium-dependent, flow-mediated dilation (FMD), and in plasma levels of thiobarbituric acid-reactive substances (TBARS) and soluble intercellular adhesion molecule-1 (sICAM-1). In 16 claudicants, maximal exercise reduced FMD (from 8.5+/-0.9 to 3.7+/-0.8%, P<0.01), and increased plasma levels of TBARS (from 1.93+/-0.06 to 2.22+/-0.1 nmol/ml, P<0.02) and of sICAM-1 (from 282+/-17 to 323+/-19 ng/ml, P<0.01). In eight of these patients, randomized to vitamin C, exercise-induced changes in FMD and biochemistry were abolished. This beneficial effect was not observed in the eight patients randomized to saline. In 15 patients, who walked until the onset of claudication pain (submaximal exercise), and in ten control subjects, who performed maximal exercise, no changes were observed with exercise. Thus, in claudicants, vitamin C prevents the acute, systemic impairment in endothelial function induced by maximal exercise. This finding provides a rationale for trials investigating antioxidant therapy and cardiovascular risk in patients with intermittent claudication.

Platelet Activation by Superoxide Anion and Hydroxyl Radicals Intrinsically Generated by Platelets That Had Undergone Anoxia and Then Reoxygenated

BACKGROUND Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation. METHODS AND RESULTS Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (-74%, P < .005), catalase (-67%. P < .005). SOD plus catalase (-82%, P < .005), and the hydroxyl radical (OH0) scavengers mannitol (-66%, P < .005) and deoxyribose (-55%, P < .005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (0-2), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2.3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH0. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin: by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium an inhibitor of flavoprotein-dependent enzymes: and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase. CONCLUSIONS This study shows that platelets exposed to A-R intrinsically generated 0-2 and OH0, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.

Measurement of oxysterols and α-tocopherol in plasma and tissue samples as indices of oxidant stress status

Oxidant stress seems to play a role in several setting of human pathology, such as atherosclerosis, cancer, and aging. The study of oxidant stress in human disease should be based on the evaluation of either sensitive and specific markers of enhanced oxidant stress, such as oxysterols, or antioxidant defense, by measuring alpha-tocopherol. We have developed a rapid method to measure the oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol in plasma (50 healthy subjects) and tissue as an index of oxidant stress in vivo, and from the same sample alpha-tocopherol content. The mean plasma concentration of 7beta-hydroxycholesterol and 7-ketocholesterol was 4.6+/-1.1 and 13.4+/-7.6 ng/mL, respectively. Plasma alpha-tocopherol concentration was 5.8+/-1.0 micromol/mol cholesterol. Samples from atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more alpha-tocopherol compared to normal arteries. No significant difference in cholesterol and oxysterol content was observed between cirrhotic and normal liver. However, cirrhotic liver contained significantly smaller concentration of alpha-tocopherol compared to normal liver. In conclusion, we have developed a rapid and reliable method for the assay of cholesterol oxidation products and alpha-tocopherol in plasma and tissue useful for estimation of oxidant stress/antioxidant balance.

Angioplasty increases coronary sinus F2-isoprostane formation: evidence for in vivo oxidative stress during PTCA

OBJECTIVES Isoprostanes, stable end-products of oxygen free radical mediated-lipid peroxidation, were measured in the coronary vessels during percutaneous transluminal coronary angioplasty (PTCA) to provide direct evidence for enhanced oxidative stress in a local milieu in vivo. BACKGROUND Percutaneous transluminal coronary angioplasty is associated with complications such as myocardial stunning and accelerated restenosis, which at least in part are mediated by oxygen free radicals. Because isoprostanes are markers of oxidant stress and potent vasoactive compounds, the formation of which is not inhibited by aspirin treatment in vivo, it is possible that these mediators are increased locally during PTCA. METHODS In 12 coronary artery disease patients who were given aspirin and ticlopidine, blood samples from coronary sinus were taken immediately before and immediately upon balloon deflation during PTCA. Isoprostane F2alpha-III, isoprostane F2alpha-VI, and TxB2 were quantified after extraction and chromatography using a stable dilution isotope gas chromatography/mass spectrometry assay. RESULTS Coronary sinus and left main coronary artery levels of iPF2alpha-III and iPF2alpha-VI at baseline were (mean +/- SEM) 40 +/- 9 pg/ml and 115 +/- 10 pg/ml, respectively. The TxB2 levels were undetectable. Following PTCA, isoprostane levels markedly increased (mean +/- SEM): iPF2alpha-III, 125 +/- 12 pg/ml (p < 0.001); iPF2alpha-VI, 295 +/- 20 pg/ml (p < 0.001), whereas TxB2 levels remained undetectable. CONCLUSIONS These results indicate that PTCA induces coronary sinus increase in F2-isoprostane formation, and they also provide direct evidence for enhanced oxidative stress in a local milieu in vivo. Thus, an increased F2-isoprostane formation could play a role in the pathogenesis of some PTCA-associated untoward events.

Iron-Dependent Human Platelet Activation and Hydroxyl Radical Formation Involvement of Protein Kinase C

BACKGROUND Iron is an important modulator of lipid peroxidation, and its levels have been associated with the progression of atherosclerosis. Little is known about the possibility that this metal, when released from tissue stores, may modulate the reactivity of blood cell components, in particular platelets. Therefore, we investigated a possible link between iron, oxygen free radical formation, and platelet function. METHODS AND RESULTS Human whole blood was stimulated with collagen 2 micrograms/mL, and an irreversible aggregation with thromboxane (Tx)B2 formation was observed (15+/-4 versus 130+/-10 ng/mL). Deferoxamine (DSF), a specific iron chelator, and catalase, an H2O2 scavenger, inhibited collagen-induced whole-blood aggregation. The aggregation was accompanied by an increase in hydroxyl radical (OH.) levels (30+/-8 versus 205+/-20 nmol/L dihydroxybenzoates), which were reduced by DSF and by 2 specific OH. scavengers, mannitol and deoxyribose. Iron (Fe2+) dose-dependently induced platelet aggregation, TxB2 formation (6+/-2 versus 135+/-8 ng/mL), and protein kinase C (PKC) translocation from the cytosol to the cell membrane when added to platelets that have been primed with a low concentration of collagen (0.2 micrograms/mL). In the same system, an increase in OH. levels was observed (37+/-12 versus 230+/-20 nmol/L dihydroxybenzoates). Mannitol and deoxyribose, but not urea, were able to reduce OH. formation, PKC activation, and platelet aggregation. Selective inhibition of PKC activity by GF 109203X prevented iron-dependent platelet aggregation without influencing OH. production. CONCLUSIONS The present study shows that iron can directly interact with human platelets, resulting in their activation. Its action is mediated by OH. formation and involves PKC activity. Our findings provide an additional contribution to the understanding of the mechanism(s) by which iron overload might promote atherosclerosis and coronary artery disease.

Oxidative stress and antioxidant therapy in cystic fibrosis.

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

A potent chain-breaking antioxidant activity of the cardiovascular drug dipyridamole

The antioxidant properties of the antithrombotic drug dipyridamole have been studied using lipid oxidation assays based on the generation of peroxy radicals by azo compounds. Dipyridamole was observed to prevent both peroxidation of arachidonic acid micelles in aqueous solution and peroxidation of methyl linoleate in organic solvents; in contrast to vitamin E, dipyridamole was found to scavenge both hydrophilic and hydrophobic radicals. The rate constant for the reaction of dipyridamole with methyl linoleate peroxyl radicals at 37 degrees C was calculated as 2 x 10(6) M-1s-1, in comparison to 1 x 10(6) M-1s-1 of vitamin E under the same conditions. The antioxidant efficiency of the drug was confirmed in experiments with radiolysis-induced oxidation and through measurements of malondialdehyde production and diene formation. As a result of radical scavenging, a relatively stable dipyridamole radical was formed that could be detected by electron spin resonance spectroscopy. The particular antioxidant properties of dipyridamole may explain the vasodilating and antiplatelet effects of this cardiovascular drug.

Radiolabeled Native Low-Density Lipoprotein Injected Into Patients With Carotid Stenosis Accumulates in Macrophages of Atherosclerotic Plaque Effect of Vitamin E Supplementation

BACKGROUND Accumulation of LDL within the arterial wall appears to play a crucial role in the initiation and progression of atherosclerotic plaque. The dynamic sequence of this event has not been fully elucidated in humans. METHODS AND RESULTS In 7 patients with previous transient ischemic attack or stroke and critical (>70%) carotid stenosis, autologous native [(125)I]-labeled LDL or [(125)I]-labeled human serum albumin were injected 24 to 72 hours before endarterectomy. Carotid specimens obtained at endarterectomy were analyzed by autoradiography and immunohistochemistry. Autoradiographic study showed that LDL was localized prevalently in the foam cells of atherosclerotic plaques, whereas the accumulation in the lipid core was negligible. Immunohistochemistry revealed that foam cells that had accumulated radiolabeled LDL were mostly CD68 positive, whereas a small number were alpha-actin positive. No accumulation of the radiotracer was detected in atherosclerotic plaques after injection of radiolabeled human serum albumin. In 3 patients treated for 4 weeks with vitamin E (900 mg/d), an almost complete suppression of radiolabeled LDL uptake by macrophages was observed. CONCLUSIONS This study shows that circulating LDL rapidly accumulates in human atherosclerotic plaque. The prevalent accumulation of LDL by macrophages provides strong support to the hypothesis that these cells play a crucial role in the pathogenesis of atherosclerosis.