Abstract: Oxidative stress, which contributes to neuronal damage, is thought to be a pathophysiological mechanism of Alzheimer's disease (AD). Markers of oxidative stress may appear early in the preclinical, mild cognitive impairment (MCI) phase of AD. We investigated the interaction among enzymatic-derived oxysterols (24S-hydroxycholesterol and 27-hydroxycholesterol), markers of oxidative stress, including free radical-related oxysterols (7beta-hydroxycholesterol and 7-ketocholesterol), and vitamin E in AD patients and two amnestic MCI subtypes, amnestic single-domain MCI (a-MCI) subjects, and multi-domain MCI (md-MCI) subjects, compared to healthy control subjects (HC). The study included 37 patients with AD, 24 with a-MCI, 29 with md-MCI, and 24 HC. Plasma assessments were made using isotope dilution-mass spectrometry. Although we found no significant change in free radical- or enzymatic-derived oxysterol concentrations in AD or MCI patients, vitamin E levels corrected for cholesterol were reduced in AD patients compared to HC. Results suggest that AD patients have upregulated oxidative cerebral stress or a nutritional deficit of vitamin E. The oxysterols investigated here are not useful markers for diagnosing AD or MCI.
Abstract: Statins and other lipid lowering drugs have been repeatedly described to decrease blood levels of minor fat soluble components such as vitamin E (as alpha-tocopherol). Clinical consequences of this secondary state of deficiency have not been described so far, but recent biochemical and molecular evidence on homeostatic and molecular responses to vitamin E deficiency in skeletal muscle cells may suggest the hypothesis presented in this paper of a role as risk factor in the development of statin-associated myopathy. This hypothesis that needs to be further investigated, could suggest the need for precautionary measures during lipid lowering therapy, which include timely diagnosis and active prevention of vitamin E deficiency.
Abstract: Enhanced oxidative stress is a common feature of liver diseases and contributes to chronic liver disease (CLD) progression by inducing fibrogenesis during liver regeneration. Peroxidation products of cholesterol metabolism, named oxysterols, are new and reliable markers of oxidative stress in vivo. Patients affected by CLDs present high plasma levels of oxysterols, raising the question of the origin and biological relevance of these compounds in the pathophysiology of chronic liver damage. The aim of this study was to examine the molecular basis of the biological effects of oxysterols on liver-derived cells, HepG2 and Huh7. Cells were treated with different concentrations (10(-9) to 10(-5) M) of 7-ketocholesterol used as a reference, and 5,6-secosterol, a recently discovered oxysterol. FACS investigations, caspase-3 activation, and Sytox Green immunofluorescent assay showed that pathological concentrations of oxysterols induced necrosis (30-50%) after 48 h of treatment. The two analyzed compounds displayed a similar, but not identical, behavior. In fact, 5,6-secosterol, but not 7-ketocholesterol, induced cell senescence. Notably, low concentrations of 5,6-secosterol caused a sustained activation of ERK1/2, inducing cell proliferation, this unexpected behavior should be better characterized by further studies. Since enhanced oxidative stress is known to worsen liver chronic hepatitis and frequently results in overall decreased cellular survival, our data suggest the important and different role oxysterols may have in interfering with physiological liver tissue regeneration in injured human liver. Antioxidant treatment may provide a highly specific and effective mean to counteract the common consequences of oxidative stress on chronic hepatitis, such as fibrosis/cirrhosis and liver failure.
Abstract: ABSTRACT: INTRODUCTION: Although acute myocardial infarction is generally associated with obstructive coronary artery disease, myocardial infarction associated with normal coronary arteries is a well-known condition. The overall prevalence rate of myocardial infarction with normal coronary arteries is considered to be low, varying from 1% to12% depending on the definition of "normal" coronary arteries. CASE PRESENTATION: We describe here a case of a 49-year-old woman with a history of prior myocardial infarction who continued to be asymptomatic after a 10-year follow-up, in the absence of a high-risk profile for adverse outcomes. She was studied with multi-slice coronary computed tomography and whole-body angiography, which showed normal coronary and extra-coronary arteries. CONCLUSION: This case report raises two important issues. First, the possible role of multi-slice computed tomography/coronary angiography in the risk- and prognosis assessment of patients with known or suspected coronary artery disease. Second, the important role played by long-term pharmacological therapy in patients with prior myocardial infarction and normal coronary arteries.
Abstract: A comprehensive whole-body approach to noninvasive evaluation of coronary and extracoronary vasculature is currently not available. The objective of our study was to assess the potential of 64-slice computed tomography angiography (64-CTA) for whole-body evaluation of atherosclerosis burden. Seventy-eight patients referred for coronary imaging underwent whole-body 64-CTA using an adjusted strategy for the administration of contrast medium with dose-saving algorithms involving ECG modulation and reduced tube voltage. Arterial segments (15 coronary, 32 systemic) were evaluated for significant (> or =50%) steno-occlusive disease while arterovenous density was evaluated at seven extracoronary locations. Homogeneous attenuation (mean 321 +/- 20 HU) was obtained throughout the systemic vasculature. Atherosclerosis was observed in 238/995 (24%) coronary and 368/2441 (15%) systemic segments. Significant stenoses/occlusions were present in 214 (21%)/24 (2.5%) coronary segments while asymptomatic clinically relevant stenoses were present in 49 systemic segments. Sensitivity, specificity, positive and negative predictive values of coronary 64-CTA among 52 patients who also underwent quantitative coronary angiography were 92%, 95%, 81% and 98%, respectively. ECG modulation decreased radiation exposure to 14.1-15.4 mSv per patient. Comprehensive, noninvasive assessment of atherosclerosis can be performed by whole-body 64-CTA and may have a positive impact on secondary prevention.
Abstract: BACKGROUND: Type 2 diabetes mellitus (T2DM) is commonly associated with both microvascular and macrovascular complications and a strong correlation exists between glycaemic control and the incidence and progression of vascular complications. Pioglitazone, a Peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand indicated for therapy of type T2DM, induces vascular effects that seem to occur independently of glucose lowering. METHODS: By using a hindlimb ischemia murine model, in this study we have found that pioglitazone restores the blood flow recovery and capillary density in ischemic muscle of diabetic mice and that this process is associated with increased expression of Vascular Endothelial Growth Factor (VEGF). Importantly, these beneficial effects are abrogated when endogenous Akt is inhibited; furthermore, the direct activation of PPARgamma, with its selective agonist GW1929, does not restore blood flow recovery and capillary density. Finally, an important collateral vessel growth is obtained with combined treatment with pioglitazone and selective PPARgamma inhibitor GW9662. CONCLUSION: These data demonstrate that Akt-VEGF pathway is essential for ischemia-induced angiogenic effect of pioglitazone and that pioglitazone exerts this effect via a PPARgamma independent manner.
Abstract: Atrial fibrillation is the most common arrhythmia in clinical practice, may coexist with conditions common to both cardiovascular and noncardiovascular diseases and is associated with considerable morbidity and mortality. Atrial fibrillation is often asymptomatic and diagnosed only when it has caused a potentially serious complication, such as an ischemic stroke. When atrial fibrillation has been identified, 2 objectives have to be addressed--the antiarrhythmic therapy based on rate control or rhythm control, and prevention of thromboembolism. A rhythm or rate control strategy can be chosen indifferently because they have comparable efficacy for the outcome measure of mortality, but the antithrombotic therapy is ever mandatory. The risk of stroke increases cumulatively with increasing age, previous transient ischemic attack or stroke, hypertension, diabetes mellitus, impaired left ventricular function and heart failure. Warfarin reduces the risk of stroke by about two thirds; and aspirin, by about one fifth, but its use must be weighted with the risk of bleeding. The risk of anticoagulant-associated hemorrhage increases with age, the presence of serious concomitant diseases, with poorly controlled hypertension and poorly controlled anticoagulation.
Abstract: BACKGROUND: Disarrangement in fatty acids and oxidative stress are features of cystic fibrosis. Cholesterol is very sensitive to oxidative stress. OBJECTIVES: The objectives were to examine whether cholesterol oxidation products are altered in cystic fibrosis and whether they are associated with fatty acids and with characteristics of the disease state. DESIGN: 7-Ketocholesterol and 7beta-hydroxycholesterol (prototype molecules of free radical-mediated cholesterol oxidation) and the fatty acid profile were assessed by mass spectrometry in patients and in sex- and age-matched control subjects. RESULTS: In a comparison with control subjects, mean (+/-SD) cholesterol oxidation was higher (7-ketocholesterol: 11.31 +/- 5.1 compared with 8.33 +/- 5.5 ng/mL, P = 0.03; 7beta-hydroxycholesterol: 14.5 +/- 6.8 compared with 9.7 +/- 4.1 ng/mL, P = 0.004), total saturated fatty acids were higher (31.90 +/- 1.93% compared with 30.31 +/- 0.98%, P < 0.001), monounsaturated fatty acids were higher (29.14 +/- 3.85% compared with 25.88 +/- 2.94%, P = 0.004), omega-6 (n-6) polyunsaturated fatty acids were lower (34.84 +/- 4.77 compared with 39.68 +/- 2.98%, P < 0.0001), and omega-3 (n-3) polyunsaturated fatty acids were comparable in patients with cystic fibrosis. Oxysterols were inversely associated with 24:0 and 18:2 omega-6 fatty acids but did not correlate with the increased oleic acid or with any of the omega-3 fatty acids. CONCLUSIONS: Cystic fibrosis is characterized by relevant cholesterol oxidation that is associated with an abnormal fatty acid profile. The interplay between oxysterols and fatty acids potentially provides insight into the biological mechanisms that underlie this complex disease.
Abstract: OBJECTIVE: To evaluate dose reduction and image quality in coronary 64-slice multidetector computed tomography using an automatic exposure control system (AECs). METHODS: A total of 101 patients were divided into 4 groups. Tube current was 600 and 800 mAs in groups A and B and adapted at 600 and 800 quality-reference mAs using an AECs in groups C and D. Effective dose and organ-equivalent dose were evaluated. Image noise was quantified as standard deviation of air-space attenuation. Two observers assessed technical adequacy and image quality using a 4-point scale. RESULTS: Effective dose ranged from 8.6 mSv (group C) to 15 mSv (group B) with significant dose reduction for examinations performed at 600 mAs (21.7%) and 800 mAs (29.4%). Contribution of organ-equivalent doses showed higher exposure for lungs (42%) and breast (22%). Noise was significantly higher in groups studied with AECs. Larger coronary segments resulted in higher image quality scores without differences between groups. CONCLUSION: Automatic exposure control systems provides images of diagnostic quality with substantial dose reduction.
Abstract: Familial combined hyperlipidemia (FCHL), the most common inherited disorder of lipid metabolism, is associated with an increased risk of atherosclerosis that is not fully explained by the metabolic disturbances of these patients. Oxidative damage to lipid components accumulating in the plasma of FCHL patients might contribute to explaining this lack of evidence. Cholesterol is one of the preferential targets of oxidation in LDL and this may contribute to setting a proatherogenetic phenotype in FCHL. We investigated plasma oxysterols (7-ketocholesterol and 7beta-hydroxycholesterol) and alpha-tocopherol as in vivo hallmarks of lipid-related oxidative stress. Oxidative stress hallmarks were measured in 45 FCHL patients and 54 sex- and age-matched healthy controls; in FCHL patients, oxidative stress and lipid profile parameters were also assessed in response to lipid-lowering drugs in a 24-week randomized, open-label trial with atorvastatin or fenofibrate. FCHL patients showed markedly increased levels of oxysterols (p < 0.001) and reduced alpha-tocopherol/total lipids (p < 0.001) compared to controls. These differences were independent of the presence of clinical atherosclerosis and persisted after correction for hyperlipidemia. Atorvastatin and fenofibrate significantly improved the lipid profile and caused a comparable decrease in plasma oxysterols, with the normalization of 7-ketocholesterol and a significant reduction of 7beta-hydroxycholesterol (p < 0.001). These drugs also decreased the ratio of alpha-tocopherol/total lipids by more than 30% (p < 0.001). In conclusion, FCHL patients showed increased hallmarks of cholesterol oxidation and decreased levels of alpha-tocopherol/total lipids. Atorvastatin and fenofibrate displayed comparable efficiency in decreasing oxysterols, but they further decreased lipid-corrected alpha-tocopherol levels in plasma. More research work is needed to understand the clinical meaning of these findings, which may help to understand the role of oxidative stress in FCHL and lipid-lowering therapy.
Abstract: The oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in patients with atherosclerosis, 7beta-hydroxycholesterol was found to be the strongest predictor of progression of carotid atherosclerosis. Interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in vitro has been reported recently, using recombinant 11beta-hydroxysteroid dehydrogenase or rodent liver microsomes. In this study deuterium-labeled 7beta-hydroxycholesterol or 7-ketocholesterol was administered intravenously to two healthy volunteers and blood samples were collected at different time points. The mean half-life for elimination of 7beta-hydroxycholesterol from the circulation was estimated to be 1.9 h. The corresponding half-life for 7-ketocholesterol was estimated to be 1.5 h. Infusion of deuterium-labeled 7-ketocholesterol resulted in labeling of 7beta-hydroxycholesterol and vice versa. In addition, the biological within-day and between-day variations of the two oxysterols were determined. In summary, the present investigation clearly shows an interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in humans.
Abstract: BACKGROUND AND OBJECTIVES: Oxysterols are markers of oxidative stress, levels of which have not yet been reported in hemodialysis (HD) patients. This study was designed to compare levels of the oxysterols 7-ketocholesterol (7KC) and 7beta-hydroxycholesterol (7betaOH) between a cohort of HD patients and healthy controls. METHODS: This nested cross-sectional study reflects baseline (pre-intervention) values for markers of oxidative stress, inflammation and nutrition status in the 160-member vitamin E and carotid intima media thickness progression in end-stage renal disease (VIPER) cohort (age 64.1 +/- 8.8, 33.5% female). Age- and sex-matched healthy volunteers served as controls. Plasma oxysterols 7KC and 7betaOH were determined by isotope dilution gas chromatography/mass spectrometry. RESULTS: Despite higher plasma alpha-tocopherol levels in HD patients than controls (36.0 +/- 9.3 vs. 31.8 +/- 8.4 micromol/l, p = 0.007), 7KC levels (9.8 +/- 6.9 vs. 5.9 +/- 2.8 nmol/mmol cholesterol, p < 0.0001) and 7betaOH levels (8.7 +/- 4.3 vs. 2.7 +/- 1.6 nmol/mmol cholesterol, p < 0.0001) were higher in HD patients. The oxysterol 7betaOH was significantly, inversely associated with prealbumin (r = -0.18, p = 0.03), though neither oxysterol was significantly associated with any other marker of oxidative stress, inflammation or nutrition status and did not discriminate for CVD in HD patients. CONCLUSIONS: Elevated levels of the oxysterols 7KC and 7betaOH indicate that HD patients are in a state of oxidative stress compared to healthy controls. However, oxysterols 7KC and 7betaOH did not appear to contribute additional information about oxidative stress among HD patients.
Abstract: Oxidative stress is implicated in the pathogenesis of hepatic ischemia-reperfusion injury, a major determinant of initial poor graft function (IPGF) after orthotopic liver transplantation (OLT). We prospectively investigated the association between the recipient plasma preoperative oxidative stress and the occurrence of IPGF after deceased-donor OLT and indirectly studied the source-hepatic or extra-hepatic-of systemic oxidative stress in vivo in cirrhosis. We used a recently developed specific and sensitive mass spectrometry assay to measure 7beta-hydroxycholesterol and 7-ketocholesterol (oxysterols), markers of oxidative stress, in biological matrices. At univariate analysis, preoperative recipient 7beta-hydroxycholesterol plasma concentration was significantly higher in transplants with subsequent IPGF (n = 9) compared with those with initial good graft function (IGGF; n = 23) [mean +/- SD: 30.63 +/- 26.42 and 11.57 +/- 15.76 ng/mL, respectively] (P = 0.017). In a logistic regression model, which included also the Model for End-Stage Liver Disease (MELD) score, 7beta-hydroxycholesterol plasma concentration was an independent predictor of IPGF with an odds ratio of 1.17 (95% CI, 1.02-1.33, P = 0.028). Patients with cirrhosis (n = 32) had increased oxysterol plasma levels compared with healthy controls (n = 49); livers with cirrhosis (n = 21), however, had oxysterol content comparable with normal livers obtained from organ donors (n = 19). Oxysterols persisted elevated in plasma 1 month after OLT (n = 23). In conclusion, cirrhosis presents upregulated systemic oxidative stress likely of extrahepatic source that is associated with graft failure after OLT.
Abstract: OBJECTIVE: Oxidative stress is believed to play a pivotal role in the initiation and progression of atherosclerosis. We analyzed whether vitamin E supplementation influences oxidative stress in plasma and atherosclerotic plaques of patients with severe atherosclerosis. METHODS AND RESULTS: In 16 patients who were candidates for carotid endarterectomy and in 32 age- and sex-matched controls, plasma levels of 7beta-hydroxycholesterol, 7-ketocholesterol, cholesterol, and vitamin E were measured. Patients were randomly allocated to standard treatment with or without 900 mg/d vitamin E. After 6 weeks of treatment, the reported variables were measured in plasma and plaques. The plasma vitamin E/cholesterol ratio was significantly lower in patients than in controls (3.05+/-0.6 versus 6.3+/-1.7 micromol/mmol cholesterol, P<0.001). Plasma 7beta-hydroxycholesterol was significantly higher in patients than in controls (5.0+/-1.04 versus 4.4+/-0.6 ng/mL, P<0.05). Patients who were given vitamin E supplementation showed a significant increase of plasma vitamin E with concomitant decrease of 7beta-hydroxycholesterol. Conversely, no treatment dependence was observed in oxysterol or vitamin E content of plaques. CONCLUSIONS: An imbalance between oxidative stress and antioxidant status is present in patients with advanced atherosclerosis. Vitamin E supplementation improves this imbalance in plasma but not in plaques.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Clinical trials with vitamin E have yielded contrasting results. In these trials, the amount of vitamin E given was different, and the compliance was not assessed in all studies. In addition, the modality of intake, ie, in relation to food, was not specified in any trial. Vitamin E is lipophilic, and its absorption is expected to be increased by food. We studied the bioavailability of vitamin E in relation to food intake and the effect on the lipid peroxide-scavenging activity of plasma and on 7beta-hydroxycholesterol and 7-ketocholesterol (oxysterols) as markers of oxidant stress. Twenty healthy Italian subjects were randomly assigned to take vitamin E at 300 mg/d on an empty stomach (group A) or during dinner (group B) for 15 days. Plasma vitamin E markedly increased in group B (84%) compared with group A (29%). The lipid peroxide-scavenging activity of plasma increased significantly in group B (14%, P=0.005) but did not change in group A. All subjects showed very low levels of plasma oxysterols, which were not affected by vitamin E supplementation in either group. This study shows that plasma concentration of vitamin E and plasma antioxidant activity in response to oral supplementation are markedly affected by food intake. Healthy Italian subjects show very low levels of cholesterol oxidation products; these low levels are possibly related to the Mediterranean diet. To obtain maximal absorption, vitamin E must be given at meals. These data should be taken into account in clinical trials with vitamin E.
Abstract: Free radical mediated oxidation of low-density lipoproteins (LDL), which has been extensively studied in the last two decades, plays a central role in the development of the atherosclerotic plaque. Oxidation involves the lipid moiety of LDL in a chain reaction mechanism. In the initial phase, free radicals preferentially attack highly oxidizable polyunsaturated fatty acids. Subsequent recruitment of other molecules includes cholesterol and phospholipids. The process of oxidation is counteracted by antioxidants present in LDL. By-products formed during oxidation of LDL lipids, which may have biological activity, react with amino acid residues of the LDL protein backbone with the consequent modification of chemical and immunological properties responsible for cellular receptor shift. Oxidation-altered apolipoprotein B of oxidized LDL is, in fact, recognized by the macrophage scavenger receptor responsible for foam cell formation. The mechanism of LDL oxidation and the impact on atherogenesis are discussed.
Abstract: Atherosclerosis is the commonest lesion of blood vessels and is responsible for life-threatening events such as myocardial infarction and stroke. In the last two decades a series of excellent studies unraveled biochemical mechanisms that provided the background for a theory of atherogenesis. This theory is centered on foam cells and on free radical-mediated modification of low density lipoprotein (LDL). Foam cells are the main cell type of atherosclerotic lesions and originate from monocytes migrated from blood and from smooth muscle cells of the arterial wall. Foam cells are engulfed of lipids taken from LDL. Paradoxically, accumulation of LDL in developing foam cells does not occur via the classic LDL receptor. Incubation of macrophages with even very high concentrations of LDL does not appreciably increase cholesterol content. Chemically modified LDL easily enter the cells of atherosclerotic plaque via an unregulated receptor, the scavenger receptor. The most studied chemical modification of LDL is that induced by free radicals.
Abstract: Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239-247) with an aromatic ring system responsible for an intense absorption band in the 400-480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7-8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N, N'-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method.
Abstract: 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.
Abstract: Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A2, inhibited collagen-induced platelet O2- and OH(0) release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH and O2- respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O2- and OH formation, which is dependent upon AA release and metabolism.
Abstract: Hydatidosis or echinococcosis is a parasitic disease caused by Echinococcus granulosus or E. multilocularis, which forms cysts in the liver and lung after penetrating the duodenal mucosa and entering the portal circulation. The liver and lung act as a filter but some embryos enter the general circulation and disseminate throughout the body. Musculoskeletal involvement is a rare manifestation of hydatidosis, which is usually reported to affect a single muscle. We report here a rare case of a 68-year-old man with widespread hydatidosis of the retroperitoneum and the subcutaneous adipose tissue, and with multiple muscle involvement in the absence of liver, lung, and spleen involvement. The patient underwent surgical excision of a subcutaneous hydatid cyst 7 years earlier. It is likely that the large dissemination of parasites resulted from accidental rupture of the primary focus during surgery with consequent release and spreading of scolices via lymphatics.
Abstract: Postmortem and in vitro studies have shown that oxidative stress plays a role in the pathogenesis of many of the clinical features of Down's syndrome. The isoprostane 8,12-iso-iPF2alpha-VI is a specific marker of lipid peroxidation. We found elevated levels of this isoprostane in urine samples of subjects with Down's syndrome compared with those of matched controls, which correlated with the duration of the disease. These results suggest that increased in vivo lipid peroxidation is a prominent component early in the course of Down's syndrome.
Abstract: Clotting activation may occur in liver cirrhosis, but the pathophysiological mechanism has not been fully elucidated. Because a previous study demonstrated that lipid peroxidation is increased in cirrhosis, we analyzed whether there is a relationship between lipid peroxidation and clotting activation. Thirty cirrhotic patients (19 men and 11 women; age, 34 to 79 years) and 30 controls matched for sex and age were investigated. In all subjects, monocyte expression of tissue factor (TF) antigen and activity; plasma levels of prothrombin fragment 1+2 (F1+2), a marker of thrombin generation; and urinary excretion of Isoprostane-F2alpha-III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5) or standard therapy plus 300 mg vitamin E twice daily (n = 9). In addition, we analyzed in vitro if vitamin E (50 micromol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2alpha-III (P <. 0001), F1+2 (P <.0001), and monocyte TF antigen (P <.0001) and activity (P <.03) than controls. Isoprostane-F2alpha-III was significantly correlated with F1+2 (Rho = 0.85; P <.0001) and TF antigen (Rho = 0.95; P <.0001) and activity (Rho = 0.94; P <.0001). After vitamin E treatment, Isoprostane-F2alpha-III (P =.008), F1+2 (P <.008), and monocyte TF antigen (P =.012) and activity (P =.008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In vitro, vitamin E significantly reduced the expression of monocyte TF antigen (-52%; P =.001) and activity (-55%; P =.003), as well as F1+2 generation (-51%; P =.025). This study shows that vitamin E reduces both lipid peroxidation and clotting activation and suggests that lipid peroxidation may be an important mediator of clotting activation in liver cirrhosis.
Abstract: The oxidative modification of low density lipoprotein (LDL) is thought to be an important factor in the initiation and development of atherosclerosis. Antioxidants have been shown to protect LDL from oxidation and to inhibit atherosclerosis development in animals. Potent synthetic antioxidants are currently being tested, but they are not necessarily safe for human use. We here characterize the antioxidant activity of IRFI005, the active metabolite of Raxofelast (IRFI0016) that is a novel synthetic analog of vitamin E under clinical development, and demonstrate that it prevents oxidative modification of LDL. IFI005 inhibited the oxidative modification of LDL, measured through the generation of MDA, electrophoretic mobility and apo B100 fluorescence. During the oxidation process IRF1005 was consumed with the formation of the benzoquinone oxidation product. The powerful antioxidant activity of IRFI005 is at least in part mediated by a chain breaking mechanism as it is an efficient peroxyl radical scavenger with a rate constant k(IRFI005 + LOO(o)) of 1.8 X 10(6) M(-1)s(-1). 4. IRFI005 substantially preserved LDL-associated antioxidants, alpha-tocopherol and carotenoids, and when co-incubated with physiologic levels of ascorbate provoked a synergistic inhibition of LDL oxidation. Also the co-incubation of IRFI005 with Trolox caused a synergistic effect, and a lag phase in the formation of the trolox-benzoquinone oxidation product. A synergistic inhibition of lipid peroxidation was also demonstrated by co-incubating IRFI005 and alpha-tocopherol incorporated in linoleic acid micelles. These data strongly suggest that IRFI005 can operate by a recycling mechanism similar to the vitamin E/ascorbate sysem.
Abstract: We measured the urinary excretion of Isoprostane F2alpha-III and Isoprostane-F2alpha-VI, two markers of in vivo lipid peroxidation, and the circulating levels of the prothrombin fragment F1+2, a marker of thrombin generation, in 18 antiphospholipid antibodies-positive patients, in 18 antiphospholipid antibodies-negative patients with systemic lupus erythematosus, and in 20 healthy subjects. Furthermore, 12 patients positive for antiphospholipid antibodies were treated with (n = 7) or without (n = 5) antioxidant vitamins (vitamin E at 900 IU/d and vitamin C at 2, 000 mg/d) for 4 weeks. Compared with antiphospholipid antibodies-negative patients, antiphospholipid antibodies-positive patients had higher urinary values of Isoprostane-F2alpha-III (P =. 0001), Isoprostane-F2alpha-VI (P =.006), and plasma levels of the prothrombin fragment F1+2 (P =.0001). In antiphospholipid-positive patients, F1+2 significantly correlated with Isoprostane-F2alpha-III (Rho =.56, P =.017) and Isoprostane-F2alpha-VI (Rho =.61, P =.008). After 4 weeks of supplementation with antioxidant vitamins, we found a significant decrease in F1+2 levels (P <.005) concomitantly with a significant reduction of both Isoprostane-F2alpha-III (P =.007) and Isoprostane-F2alpha-VI (P <.005). No change of these variables was observed in patients not receiving antioxidant treatment. This study suggests that lipid peroxidation might contribute to the activation of clotting system in patients positive for antiphospholipid antibodies.
Abstract: 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.
Abstract: An augmented systemic production of thromboxane (TX) A2, as assessed by urinary excretion of the thromboxane metabolites, has been described in severe liver cirrhosis. However, the significance of this finding remains unclear since in liver cirrhosis a number of phenomena i.e. altered hepatic TXA2 metabolism, increased intrasplenic platelet destruction, may affect TXA2 entry into systemic circulation as well as its metabolism. In order to further clarify this, we measured both major enzymatic metabolites of TXB2 in the urine of 44 patients affected by liver cirrhosis, subdivided in three classes on the basis of Child-Pugh criteria. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were assayed with previously validated RIA techniques. The urinary excretion rate of 11-dehydro-TXB2 was significantly (p = 0.0001) increased in the cirrhotic patients (673.5 pg/mg cr, median) in comparison with the controls (275 pg/mg cr, median) but no significant difference could be demonstrated among the excretion rates of the three patient subgroups. The excretion rate of 2,3 dinor-TXB2 was also significantly (p = 0.0001) increased in the patients (824 pg/mg cr, median) in comparison with controls (175 pg/mg cr, median), with a significant (p < 0.05) increase from class A (381 pg/mg cr) to class C (1337 pg/mg cr). The sum of the two enzymatic metabolites was significantly (p = 0.0001 ) increased in the cirrhotic patients in comparison to controls, with a progressive increase from class A (1003 pg/mg cr, median) to class C (2240 pg/mg cr, median). The urinary excretion of 2,3 dinor-TXB2 was significantly (p = 0.008) related to plasma prothrombin fragment 1+2 (F1+2). This study provides further evidence of increased thromboxane biosynthesis in liver cirrhosis. Moreover, we demonstrate intraliver shift of thromboxane metabolic disposition, due to progressive liver decompensation, because only the fraction undergoing beta-oxidation to 2,3-dinor-TXB2 was progressively increased with the degree of liver failure. We, also, find a significant correlation between urinary excretion of 2,3-dinor-TXB2 and plasma F1+2, suggesting that clotting activation could partly account for in vivo platelet activation.
Abstract: BACKGROUND: Lipid peroxidation is thought to play a role in the evolution of liver damage, based on evidence in experimental models. However, evidence that lipid peroxidation occurs in patients with liver disease remains to be provided. We addressed the hypothesis by measuring levels of 8-epi Prostaglandin F2 alpha a bioactive prostaglandin isomer produced by free radical catalyzed peroxidation of arachidonic acid, in patients with liver cirrhosis. METHODS: In 42 patients with hepatic cirrhosis 8-epi Prostaglandin F2 alpha, factor VII activity, endotoxemia, carotenoids and alpha-tocopherol were measured. In 10 patients 8-epi Prostaglandin F2 alpha was also measured before and 30 days after 300 mg b.i.d. vitamin E administration. RESULTS: Cirrhotic patients had significant higher 8-epi Prostaglandin F2 alpha, excretion than controls [median (range): 199.2 (60.0-812) vs 85.9 (55.6-160.0) pg/mg creatinine, p < 0.0001]. Patients with urinary 8-epi Prostaglandin F2 alpha above the range in controls were more likely to have moderate or severe than mild liver failure (p < 0.004). They also had lower factor VII activity (62 +/- 19 vs 74 +/- 15%, P < 0.02) than patients with normal levels of the isoprostane. Urinary excretion of 8-epi Prostaglandin F2 alpha correlated directly with endotoxemia (Rho = 0.56, p < 0.0002) and inversely with factor VII (Rho = -0.39, p < 0.02). Cirrhotic patients given vitamin E showed a significant decrease of urinary 8-epi Prostaglandin F 2 alpha [median (range): 342.5 (170 - 812) vs 292.5 (142-562) pg/mg creatinine, p < 0.04]. CONCLUSION: This study demonstrated that lipid peroxidation is increased in vivo in patients with cirrhosis and suggests that oxidant stress might contribute to the deterioration of liver disease.
Abstract: Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2alpha may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.
Abstract: BACKGROUND AND OBJECTIVE: Prospective studies have shown that high plasma levels of fibrinogen are independently associated with the risk of cardiovascular complications. In patients suffering from peripheral vascular disease (PVD) fibrinogen has been shown to be an independent predictor of cardiovascular disease but its determinants have never been examined in this clinical setting. DESIGN AND METHODS: Fibrinogen levels were related to clinical and laboratory variables in 2,111 patients suffering from PVD. We also analyzed whether there was a regional distribution of risk factors. RESULTS: The median values of fibrinogen was 312 mg/dL. The clinical variables examined did not differentiate patients with elevated or normal fibrinogen levels. In particular, patients with ankle/arm pressure ratio < 0.8 did not show a higher prevalence of fibrinogen > 312 mg/dL. Conversely, white blood cell (WBC) count and serum cholesterol levels were significantly associated with high fibrinogen levels (p < 0.0001). Multiple logistic regression analysis demonstrated that areas of Italy were differently associated with high plasma fibrinogen levels (p < 0.03): subjects in the north and middle of Italy having significantly higher values of fibrinogen than subjects in the south of Italy (p < 0.01). A similar regional distribution was observed for WBC count and serum cholesterol levels. INTERPRETATION AND CONCLUSIONS: The regional distribution of risk factors raises the question as to whether the already reported large variability of cardiovascular events so in PVD may be attributed to a non homogeneous distribution of risk factors.
Abstract: 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.
Abstract: 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.
Abstract: The mechanism leading to the formation of antiphospholipid antibodies (aPL) is still unknown. Because an in vitro study suggested that aPL may derive from pro-oxidant conditions, we sought a relationship between aPL and isoprostanes, indices of lipid peroxidation in vivo. Thirty patients with systemic lupus erythematosus have been studied. Seventeen (56.6%) were positive for aPL because they had lupus anticoagulant and/or high titer of anticardiolipin antibodies (aCL). Plasma levels of tumor necrosis factor (TNF ) and urinary excretion of two isoprostanes, 8-epi-PGF2alpha and IPF2alpha -I, free radical catalyzed oxidation products of arachidonic acid, were measured. Patients with systemic lupus erythematosus had higher urinary excretion of 8-epi-PGF2alpha and IPF2alpha -I than controls; urinary excretion of the two isoprostanes was highly correlated (Rho = 0.74, P < .0001). Urinary 8-epi-PGF2alpha was highly correlated with both aCL titer (Rho = 0. 70, P < .0001) and TNF (Rho = 0.84, P < .0001), a measure of disease severity. Excretion of this isoprostane was also higher in those patients who exhibited aPL (P < .0001). Comparable correlations were observed with the isoprostane IPF2alpha -I. No difference of 8-epi-PGF2alpha was observed between patients with and without previous history of thrombosis. This study, showing the existence of a close association between aPL and increased in vivo lipid peroxidation, supports the hypothesis that these antibodies may result from pro-oxidative conditions and suggests that inflammation may play an important role.
Abstract: 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.
Abstract: Radiolabelled autologous low density lipoprotein (LDL) has previously been used to study in vivo distribution and metabolism of native-LDL. Non-invasive imaging of atherosclerotic lesions using 99mTc-LDL was shown to be feasible in animal models and patients but the clinical utility remains to be assessed. Since recent reports suggest that oxidized LDL may play a major role in the pathogenesis of atherosclerosis, we developed a technique to oxidize autologous LDL and compared the biodistribution of oxidized-LDL with that of native-LDL in man. In addition, we evaluated the uptake in vivo of oxidized- and native-LDL by atherosclerotic plaques. LDL, obtained from human plasma was treated with various combinations of copper ions and H2O2 to induce oxidative modification by increasing the content of lipid peroxidation products and electrophoretic mobility. When LDL (0.3 mg/ml) was incubated with 100 microM Cu2+ and 500 microM H2O2 oxidation occurred rapidly within 1 h, and was labelled with 99mTc efficiently as native LDL. In vivo distribution studies revealed a faster plasma clearance of oxidized-LDL compared to native-LDL, and a higher uptake by the reticuloendothelial system. Tomographic scintigraphy of the neck in patients suffering from transient ischemic attacks, revealed accumulation of radiolabelled LDL preparations in the carotid artery affected by atherosclerotic lesions. We developed a technique to rapidly oxidize LDL using copper and H2O2. Biodistribution data demonstrate that oxidized-LDL is rapidly cleared from circulation, is taken up mostly by organs rich in macrophages, and can be detected at the level of carotid plaques.
Abstract: 1. The oxidative modification of low density lipoprotein (LDL) is thought to be an important factor in the initiation and development of atherosclerosis. Natural and synthetic antioxidants have been shown to protect LDL from oxidation and to inhibit atherosclerosis development in animals. Synthetic antioxidants are currently being tested, by they are not necessarily safe for human use. 2. We have previously reported that dipyridamole, currently used in clinical practice, is a potent scavenger of free radicals. Thus, we tested whether dipyridamole could affect LDL oxidation at chemical and cellular level. 3. Chemically induced LDL oxidation was made by Cu(II), Cu(II) plus hydrogen peroxide or peroxyl radicals generated by thermolysis of 2,2'-azo-bis(2-amidino propane). Dipyridamole, (1-10 microM), inhibited LDL oxidation as monitored by diene formation, evolution of hydroperoxides and thiobarbituric acid reactive substances, apoprotein modification and by the fluorescence of cis-parinaric acid. 4. The physiological relevance of the antioxidant activity was validated by experiments at the cellular level where dipyridamole inhibited endothelial cell-mediated LDL oxidation, their degradation by monocytes, and cytotoxicity. 5. In comparison with ascorbic acid, alpha-tocopherol and probucol, dipyridamole was the more efficient antioxidant with the following order of activity: dipyridamole > probucol > ascorbic acid > alpha-tocopherol. The present study shows that dipyridamole inhibits oxidation of LDL at pharmacologically relevant concentrations. The inhibition of LDL oxidation is unequivocally confirmed by use of three different methods of chemical oxidation, by several methods of oxidation monitoring, and the pharmacological relevance is demonstrated by the superiority of dipyridamole over the naturally occurring antioxidants, ascorbic acid and alpha-tocopherol and the synthetic antioxidant probucol.
Abstract: 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.
Abstract: Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in non-insulin-dependent diabetic patients. For this purpose, we investigated the effect of polyol pathway inhibition on the glutathione redox status in these patients. We measured GSH and GSSG levels in erythrocytes of non-insulin-dependent diabetic patients (n = 15) before and after 1 week of treatment with placebo, followed by 1 week of treatment with an aldose reductase inhibitor (tolrestat 200 mg/dl). We found lower GSH levels (7.7 +/- 1.4 mumol/g hemoglobin [Hb]), higher GSSG levels (0.35 +/- 0.09 mumol/g Hb), and lower GSH/GSSG ratios (23.9 +/- 7.7) in diabetics compared with controls (n = 15; 9.8 +/- 0.8 mumol/g Hb, P < .001; 0.17 +/- 0.02, P < .001; and 58.3 +/- 9.1, P < .001, respectively). We did not demonstrate any statistical difference after 1 week of treatment with placebo. In contrast, the treatment with tolrestat induced a significant increase in GSH (8.9 +/- 0.7 mumol/g Hb, P < .01), a decrease in GSSG (0.25 +/- 0.06 mumol/g Hb, P < .02), and an increase in the GSH/GSSG ratio (37.3 +/- 8.4, P < .01). These data strongly support the hypothesis that the polyol pathway plays an important role in the impairment of the glutathione redox status in diabetic patients.
Abstract: Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.
Abstract: The activation of human platelets by polymorphonuclear leukocytes (PMN) was investigated in human whole blood challenged with "priming" concentrations of arachidonic acid or collagen in the presence or absence of N-formyl-Met-Leu-Phe (FMLP), a selective activator of PMN. With the use of arachidonic acid or collagen alone at priming concentrations or FMLP alone, no platelet response was observed. In contrast, FMLP in combination with arachidonic acid or collagen caused irreversible platelet aggregation with thromboxane A2 production. Platelet response to FMLP-activated PMN was enhanced by superoxide dismutase and blocked by catalase or the NADPH oxidase inhibitor diphenyliodonium, suggesting a role for the O2-.-H2O2 system in this cellular interaction. This was corroborated by experiments with exogenously added H2O2, which mimicked FMLP effects in the activation of primed platelets in whole blood. The present investigation indicates that platelets primed with minute amounts of arachidonic acid or collagen can be activated, in human whole blood, by oxygen-reactive species released by PMN.
Abstract: The interaction of a recently developed intracellular superoxide dismutase analogue, Fe(II)-N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (Fe(II)-TPEN), with reactive oxygen species was investigated under in vitro conditions. The complex catalyzed the dismutation of enzyme- or radiolysis-generated superoxide with the production of H2O2; under steady-state conditions the equilibrium was strongly shifted toward Fe(III)-TPEN. Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radicals in a Fenton reaction. The oxidized Fe(III)-TPEN was readily reduced by ascorbate or glutathione. Given the capacity to produce hydroxyl radicals and the reaction with cellular reductants it seems unlikely that Fe-TPEN may find widespread use as an intracellular superoxide dismutase substitute.
Abstract: The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen, it induced platelet aggregation and serotonin release in a dose-dependent fashion. The increase in platelet aggregation was associated with thromboxane A2 production and was prevented by 100 mumol/L aspirin. The amplification of platelet response by H2O2 was also inhibited 2 hours after 300 mg aspirin was given to healthy subjects. H2O2 alone did not affect intraplatelet Ca++ influx or mobilization but, combined with subthreshold concentrations of arachidonic acid, it increased Ca++ mobilization. In platelets prelabeled with tritiated arachidonic acid, H2O2 induced tritium release in a dose-dependent fashion; this effect was prevented by mepacrine, an inhibitor of the phospholipase A2 enzyme. Platelet function was not affected by using H2O2 in combination with other agonists such as thrombin, calcium ionophore, or adenosine diphosphate. This study suggests that H2O2 triggers activation of platelets preexposed to agonists at subthreshold levels by stimulating arachidonic acid metabolism, likely by stimulating the phospholipase A2 enzyme. The stimulation of platelets by concentrations of H2O2 similar to those released by activated leukocytes may give new insights into the functional cooperation between leukocytes and platelets.
Abstract: Dipyridamole [2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d)pyri midine], a well known platelet aggregation inhibitor, shows powerful hydroxyl radical scavenging activity by inhibiting OH.-dependent salicylate and deoxyribose degradation. Steady-state competition kinetics experiments with deoxyribose were carried out to evaluate the second-order rate constant for the reaction between hydroxyl radical and dipyridamole. OH. radicals were generated either by a Fenton-type reaction or by X-ray irradiation of water solutions. A second-order rate constant k(Dipyridamole + OH.) of 1.72 +/- 0.11 X 10(10) M-1 s-1 and of 1.54 +/- 0.15 X 10(10) M-1 s-1 was measured by Fenton chemistry and by radiation chemistry, respectively. Mannitol was used as an internal standard for hydroxyl radicals in steady-state competition experiments with deoxyribose. A rate constant K(Mannitol + OH.) of 1.58 +/- 0.13 X 10(9) M-1 s-1 and 1.88 +/- 0.14 X 10(9) M-1 s-1 was measured in the Fenton model and in the water radiolysis system, respectively. Both these rate constants are in good agreement with the published data obtained by the "deoxyribose assay" and by pulse radiolysis.
Abstract: It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptor-based, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation of platelets.
Abstract: In order to verify if H2O2 affects platelet function, platelet-rich plasma and human washed platelets were incubated with subthreshold concentrations (STC) of collagen or arachidonic acid or ADP and/or with 75-150 microM H2O2. While H2O2 alone did not affect platelet aggregation, it amplified platelet aggregation response in samples stimulated with STC of arachidonic acid and collagen but not in samples stimulated with STC of ADP. When platelets were preventively treated with aspirin, a cyclooxygenase inhibitor, the platelet activation by H2O2 was not observed. Thromboxane A2 (TxA2) was not produced by human washed platelets stimulated with STC of arachidonic acid, collagen or by H2O2 alone. On the contrary, when STC of agonists were tested on platelets supplemented with H2O2 an evident TxA2 production was seen. This effect was prevented by aspirin pretreatment or by the addition of catalase, an enzyme which destroys H2O2. This study suggests that H2O2 triggers the activation of platelets exposed to STC of collagen and arachidonic acid, via the cyclooxygenase pathway.
Abstract: The clinical meaning of high values of blood lipid peroxides, assessed as thiobarbituric acid reactive substances (TBA-RS), was investigated in 19 selected high risk patients with transient ischemic attacks (TIA). Patients were checked every 3-6 months and followed-up for 3 years. 8 patients experienced further vascular episodes, 4 having minor stroke and 4 TIA; one of the latter died from myocardial infarction. Unlike blood cholesterol and glucose. TBA-RS values discriminated patients with vascular episodes: they, indeed, showed significant higher values of TBA-RS. Discriminant analysis further indicated that TBA-RS levels differentiate patients with and without vascular accidents, suggesting that high blood values of lipid peroxides could represent a predictive sign of vascular ischemia.
Abstract: Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthreshold concentrations used are not able to activate platelets but "prime" platelets to be activated by SOD. The addition of SOD to arachidonic acid-or collagen-primed platelets induced aggregation, thromboxane A2 production, and release of [3H]serotonin. Superoxide dismutase does not have any effect on resting platelets and ADP-, thrombin-, calcium ionophore A23187-, PAF-, or U46619-stimulated platelets. Furthermore, superoxide dismutase-dependent platelet activation is fully prevented by catalase and/or aspirin, suggesting a role for H2O2 and the involvement of the cyclooxygenase pathway of arachidonic acid in such activation.
Abstract: This study investigates the influence of dipyridamole on platelet aggregation as evaluated by a single agonist or a pair of agonists in human platelet rich plasma and whole blood. Dipyridamole up to 30 microM was not found to influence the platelet aggregation of platelet rich plasma or whole blood; aspirin (100 microM), on the contrary, did inhibit platelet aggregation. The inhibition of platelet aggregation by aspirin could be reversed by using high concentrations of agonists or pairs of agonists. In this model dipyridamole inhibited platelet aggregation in both platelet rich plasma and whole blood in a dose-dependent fashion. Thromboxane A2 was less than 10% of controls in aspirin-treated PRP stimulated with low or high concentrations of collagen or with a pair of agonists. This study suggests that dipyridamole has direct antiplatelet activity in platelet rich plasma and whole blood when the cyclooxygenase pathway is blocked by aspirin.
Abstract: Platelet aggregation induced by threshold concentrations of agonists such as collagen, PAF or epinephrine was inhibited in vitro by 100 microM aspirin but was restored by stimulating platelets with high concentrations of collagen, PAF or by a combination of epinephrine and PAF. Incubating aspirin-treated platelets with 50-100 microM vitamin E or vitamin E acetate inhibited platelet aggregation by high concentrations of collagen and PAF and by the combination of epinephrine and PAF; platelet thromboxane A2 formation was less than 10% in samples incubated with 100 microM aspirin. Apyrase, added to aspirin-treated platelet, did not influence platelet aggregation induced by epinephrine and PAF. The present study suggests that concentrations of vitamin E as low as 50-100 microM inhibit cyclooxygenase-independent platelet aggregation when combined with an inhibitor of the arachidonate pathway.
Abstract: The influence of four hydroxil radical (OH.) scavengers on platelet function was investigated. OH. scavengers inhibited ADP, collagen, arachidonic acid, PAF-induced platelet aggregation, and platelet cyclooxygenase pathway activation, which was studied by evaluating platelet malondialdehyde and serum thromboxane A2 formation. The latter was not affected by superoxide dismutase, catalase, or metal ion chelants such as desferioxamine or DETAPAC. The detection of deoxyribose degradation by stimulated platelets suggested that platelets produce OH.. This study shows that activated platelets produce free radicals and that antioxidant agents such as OH. scavengers inhibit platelet function.
Abstract: The effect of picotamide on platelet function has been studied in vitro and ex vivo. Picotamide at micromolar concentrations inhibited platelet aggregation induced by ADP, arachidonic acid and collagen, and it also inhibited the production of thromboxane A2 (TxA2). Unlike aspirin, picotamide did not affect the synthesis of prostacyclin by blood vessels. In eight healthy subjects who took picotamide 1200 mg/d platelet aggregation and TxA2 production were inhibited. Picotamide appears to be an antiplatelet drug that reduces TxA2 synthesis without affecting cyclo-oxygenase activity.
Abstract: The study was carried out in order to evaluate if Ticlopidine induces lipid metabolism changes. Twenty seven healthy subjects were studied, 14 with placebo and 13 with Ticlopidine treatment (500 mg/day), for 30 days. Total cholesterol, HDL cholesterol, triglycerides, apolipoproteins A and B were evaluated before and after treatment. No significant changes of the blood lipid parameters were observed.
Abstract: Thrombin-induced platelet malondialdehyde (MDA) formation and plasma malondialdehyde-like material (MDA-LM) were evaluated in 12 healthy subjects before and after 1 and 7 days from aspirin (1 g) ingestion. 24 hr after aspirin administration, platelet MDA was almost abolished while MDA-LM showed a 23% decrease. Platelet MDA and plasma MDA-LM returned to baseline values 7 days after aspirin ingestion. These data suggest that platelet cyclooxygenase pathway affects only in part plasma MDA-LM. The evaluation of plasma MDA-LM before and after aspirin could be useful for evaluating in vivo platelet cyclooxygenase activation.
Abstract: A simple and rapid spectrophotometric method for evaluating platelet superoxide dismutase is reported. Platelets prepared avoiding erythrocyte and leucocyte contamination were lysated and tested in a Tris-cacodylic buffer containing pyrogallol. Platelet superoxide dismutase was calculated by evaluating the degree of the pyrogallol autoxidation inhibition induced by platelet lysate. Possible interferences of hydrogen peroxide, peroxidases and reducing substances were excluded. Platelet superoxide dismutase content was studied in 38 healthy subjects and was 19.1 +/- 4.1 U/10(8) platelets or 35 +/- 7.8 U/mg protein.
Abstract: Apolipoproteins A-I, A-II and B were evaluated in 18 chronic active hepatitis and 27 liver cirrhotic patients. The latter were also divided into compensated and decompensated subgroups. Significantly low values of apolipoproteins A-II and B were seen in chronic active hepatitis and liver cirrhotic patients, while apolipoprotein A-I was decreased in liver cirrhotic patients only. Chronic active hepatitis had higher apolipoprotein values than liver cirrhosis and in the latter one decompensated subgroup showed lower apolipoprotein levels than the compensated one. Apolipoproteins A-I, A-II and B correlated also well with prothrombin activity (Normotest) in liver cirrhotic patients, especially the apolipoprotein A-II values. This study suggests that serum values of apolipoproteins are affected by the type of liver damage and that their decrease could be partly due to impaired liver synthesis.
Abstract: The behaviour of plasma malondialdehyde-like material (MDA-LM) was evaluated in 13 healthy subjects by a single-blind study that consisted of placebo (30 days), vitamin E treatment (300 mg/day) (30 days) and placebo (30 days). The study demonstrated that MDA-LM did not change during placebo treatment while it significantly decreased after vitamin E administration.
