Laura Mazzanti

Reactive oxygen species plasmatic levels in ischemic stroke

Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion, and the antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. The aim of this study was to investigate the status of oxidative stress, NO and ONOO− levels in patients with atherothrombotic and lacunar acute ischemic stroke and iNOS, eNOS and nitrotyrosine expression in the same patients. Plasma ONOO− levels were significantly higher in patients than in controls while NO decreases in patients in respect to controls. Densitometric analysis of bands indicated that iNOS and N-Tyr protein levels were significantly higher in patients in respect to controls. This study has highlighted a significant NO decrease in our patients compared with controls and this is most probably due to the increased expression of inducible NO synthase by the effect of thrombotic attack. In fact, the constitutive NO isoforms, which produce small amounts of NO, are beneficial, while activation of the inducible isoform of NO, which produces much more NO, causes injury, being its toxicity greatly enhanced by generation of peroxynitrite. The significant ONOO− increase observed in our patients, compared to controls, is most probably due to reaction of NO with O2·−. These findings suggest that free radical production and oxidative stress in ischemic stroke might have a major role in the pathogenesis of ischemic brain injury. Peroxynitrite might be the main marker of brain damage and neurological impairment in acute ischemic stroke.

Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes

It has been recently hypothesized that in PIH a placental oxidant-antioxidant imbalance might cause the release of lipoperoxidation products into the circulation, with subsequent damage of endothelial cell membranes. In this hypothesis the endothelial cell and further increase in circulating lipoperoxide levels, which are by themselves able to induce smooth muscle constriction and increased pressor responsiveness to angiotensin II. In order to investigate this issue, we studied the basal content of lipid peroxides in terms of malondialdehyde (MDA) in the syncytiotrophoblast plasma membranes (SPM) from PIH women. Moreover, we investigated the susceptibility to peroxidation of SPM using anin vitro oxidative stress as a tool to verify the predisposition to thein vivo development of peroxidation products. The fatty acid composition of the membranes was also analyzed. Microvillus membrane lipoperoxide concentrations were significantly increased in PIH women (62.8±7.6 ng MDA/mg prot) compared with healthy pregnant subjects (37.6±4.8 ng MDA/mg prot; p<0.01).The formation of TBARS under the action of phenylhydrazine was significantly greater in PIH women (90.3±7.4 mmol MDA/mol cholesterol) than in normal pregnant subjects (68.6±6.4 mmol MDA/mol cholesterol; p<0.01). In PIH microvillus membrane we also observed a significant increase of the content of polyunsaturated arachidonic acid.The increased susceptibility to oxidative stress of SPMs from PIH women might be due either to reduced antioxidant systems or to an abnormality of the lipid composition of the membrane. The present work also demonstrated in PIH a reduction in the SPM content of saturated fatty acids with an increase in polyunsaturated fatty acids, which are the major substrate for peroxidation. On the other hand, the higher lipoperoxidation may be due to the observed increased susceptibility to peroxidative stress, to a primary reduction in placental perfusion with tissue hypoxia or to both factors, which can potentiate each other.

Modifications of platelet from Alzheimer disease patients: a possible relation between membrane properties and NO metabolites.

Alzheimer disease (AD) is a chronic neurodegenerative disorder characterised by a progressive loss of memory and cognitive functions. The formation of nitric oxide (NO), by astrocytes, has been suggested to contribute to the neurodegnerative process. Some studies have described the participation of different isoforms of NOS in the progression of AD. The present work was conducted in order to investigate the role played by NO and peroxynitrite in platelets from AD patients, the possible correlation with Na(+)/K(+)-ATPase activity and the intracellular Ca(2+) in the same group of patients as well as the expression of NOS isoenzymes and nitrotyrosine as markers of NO synthesis and reactive protein nitration. NO production was significantly elevated in the platelets from AD patients compared to controls as well as l-arginine/NO-dependent ONOO(-). Membrane Na(+)/K(+)-ATPase activity was significantly decreased in patients than in controls while intracellular Ca(2+) concentration shows an opposite trend. Platelet from AD patients showed a significantly increased 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy compared with controls. Western blot analysis using anti-iNOS and eNOS monoclonal antibodies demonstrated that both isoforms were detectable in cell lysates as well as nitrotyrosine more pronounced in the cell lysates from AD patients than controls. In conclusion, the increased expression and activity of nitrergic system may produce platelet membrane alteration or vice versa. These modifications may contribute further to the neurodegenerative process in AD because the abnormal function of Alzheimer platelets play a very important role in the pathogenesis of the disease.

Biophysical studies on agents affecting the state of membrane lipids: biochemical and pharmacological implications.

SummaryThe phospholipid requirement of membrane-bound enzymes may depend on several reasons. In our laboratory we have investigated lipids (1) as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and (2) as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins.We have found that Coenzyme Q, once reduced by NADH dehydrogenase, must cross the inner mitochondrial membrane; only quinones having long isoprenoid side chains can easily cross phospholipid bilayers, and this is the reason why a short chain quinone such as CoQ-3 inhibits NADH oxidation. The incapability of short quinones to cross lipid bilayers is due to their disposition in the lipid bilayer, stacked within the phospholipids.The conformational role of lipids has been investigated indirectly observing the kinetics of membrane-bound enzymes, e.g. the mitochondrial ATPase, and directly by circular dichroism. Lipid removal or lipid perturbation with organic solvents induce a decrease of α-helical content in mitochondrial proteins, and give rise to a series of kinetic changes in ATPase, including uncompetitive inhibition, increased activation energy, and loss of cooperativity in oligomycin inhibition.The recognition of a conformational role of lipids has allowed us to postulate a working hypothesis for the mechanism of action of general anesthetics. Such drugs have been found by us, by means of spin labels and fluorescent probes, to disrupt lipid protein interactions in several membranes, including synaptic membranes. The loosening of such interactions is believed to induce conformational changes, which will alter ion transport systems necessary to the propagation of neural impulses. Conformational changes induced by anesthetics have been found by us both directly by circular dichroism and indirectly by enzyme kinetics.The conformational effect of anesthetics is not directly exerted on the porteins but is mediated through the lipids. In agreement with this hypothesis we have found that membrane-bound acetylcholinesterase is inhibited by anesthetics, whereas the solubilized enzyme is not inhibited. However, binding of the solubilized enzyme to phospholipids restores anesthetic inhibition.

Altered platelet membrane dynamic properties in type 1 diabetes

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.

Platelet membrane fluidity and Na+/K+ ATPase activity in acute stroke

Stroke is a consequence of a reduction in cerebral blood flow but the mechanisms involved in the production of ischemic damage are complex and probably not fully known. It is hypothesized that alterations in platelet membrane fluidity are directly related to the severity of the stroke as measured by the National Institute of Health Stroke Scale (NIHSS). Thus, the aim of the present study was to investigate Na+/K+ ATPase activity and platelet membrane fluidity, measured by fluorescent probes TMA-DPH and DPH in patients affected by ischemic stroke and controls in order to identify, if any, chemical-physical and/or functional modifications associated with cerebral ischemic damage. Patients were divided into three groups according to the presence of vascular risk factors (Diabetes Mellitus, Hypertension and Smoking) in order to evaluate the possible influence of each risk factor on the NIHSS score and both Na+/K+ ATPase activity and platelet membrane fluidity. Data showed a significant decrease in both Na+/K+ ATPase activity and platelet fluidity values in patients compared to controls. Moreover, all three groups showed a negative significant correlation between NIHSS and Na+/K+ ATPase activity and a positive significant correlation between NIHSS, TMA-DPH and DPH. In conclusion, the present data point out that alterations in the platelet membranes chemical-physical (decreased fluidity) and functional properties (reduced Na+/K+ ATPase activity) rose proportionally with NIHSS increase. These modifications and their interaction with some vascular risk factors might be involved in the pathogenesis of ischemic damage development.

Reduced nitric oxide levels in acromegaly: cardiovascular implications

Acromegaly is characterized by major cardiovascular alterations. Although the underlying mechanisms of these vascular modifications have not been elucidated, recent studies have focused on endothelial dysfunction. Nitric oxide (NO) may contribute to increased vascular resistance, reduced platelet aggregation, inhibition of smooth muscle cell proliferation, and reduction of lipoxygenase activity. At present, no data on NO production in acromegalics are available. The aim of this study was to evaluate the effect of high levels of growth hormone (GH) and insulin‐like growth factor‐1 (IGF‐1) present in acromegaly on NO pathway to investigate the role played by this molecule in the cardiovascular changes experienced by these patients. We studied 13 acromegalics and 12 sex‐ and age‐matched normotensive controls. Platelet NO levels were measured in the supernatant of lysed platelets. Endothelial NO synthase (eNOS) was determined by Western blot analysis of platelets. NO concentrations were significantly reduced in patients (p<0.0001). There were no differences between male and female patients, nor were platelet NO levels and the presence/absence of hypertension related in acromegalics; by contrast, NO concentrations inversely correlated with GH (p = 0.03) and IGF‐1 (p = 0.04) levels, and with disease duration (p = 0.04). eNOS protein concentrations were significantly reduced in the platelets of patients compared with controls (p<0.0001). This study demonstrates for the first time a strong reduction in platelet NO concentrations in acromegalic patients due to reduced eNOS expression. Moreover the inverse correlation of NO levels with GH, IGF‐1 and disease duration suggests that reduced levels of platelet NO linked to GH excess may contribute to the vascular alterations affecting patients with acromegaly.

Effect of consumption of dark chocolate on oxidative stress in lipoproteins and platelets in women and in men.

The goal of this research was to investigate the effects of 3 weeks consumption of 50 g flavonoid-rich dark chocolate on lipoprotein oxidative stress in vitro and in vivo in 25 women compared to 25 men. Levels of thiobarbituric acid-reactive substances, conjugated dienes and hydroperoxide levels in HDL and LDL before and after consumption of dark chocolate were determined. Moreover in platelets of the same subjects NO and peroxynitrite levels were studied. TBARs concentration in womens HDL decreased by 26.7% while in mens HDL 23.4%; lipid hydroperoxides decreased in womens HDL by 62.8% while in mens HDL they decreased by 21.1%. Conjugate diene formation decreased in womens HDL by 55.9%, while in mens HDL it decreased by 49.2%. Moreover TBARs concentration decreased in womens LDL by 26.7% after supplementation and in mens LDL by 21.6%; lipid hydroperoxides decreased in womens LDL by 83.6% while in mens LDL they decreased by 64.7%. Moreover conjugate diene formation decreased in womens LDL by 48.2%, while in mens LDL it decreased by 21.6%. After supplementation peroxynitrite values decreased in women by 24% and in men by 18.6% while NO increased after supplementation by 15.7% compared to basal determination in women, and by 32.2% in men. This study showed that a short-term intake of dark chocolate might improve the lipoprotein profile in healthy humans, more so in women than in men, and this might exert a protective effect on the cardiovascular system.

Role of raloxifene on platelet metabolism and plasma lipids

Background  This study was performed to understand the metabolic effects of raloxifene, a selective oestrogen receptor modulator, on platelets in healthy non‐obese postmenopausal women. The data were compared to untreated subjects.

Gestational diabetes affects platelet behaviour through modified oxidative radical metabolism

Aims  Patients with Type 1 and Type 2 diabetes mellitus show altered platelet function including decreased nitric oxide synthase (NOS) activity and increased peroxynitrite production. Gestational diabetes mellitus (GDM) is a clinical condition which is ideal for evaluating short‐term effects of impaired glucose metabolism, ruling out the possibility that the platelet abnormalities are a consequence of diabetic complications. The aim of the present work was to study NO metabolism in platelets from pregnant women with GDM. The production of peroxides was also studied as it is strongly involved in peroxynitrite formation.