Laura Nanetti

Effect of homocysteinylation of low density lipoproteins on lipid peroxidation of human endothelial cells

Homocysteine‐thiolactone (HcyT) is a toxic product whose synthesis is directly proportional to plasma homocysteine (Hcy) levels. Previous studies demonstrated that the interaction between HcyT and low density lipoproteins (LDL) induces the formation of homocystamide‐LDL adducts (Hcy‐LDL). Structural and functional alterations of Hcy‐LDL have been described and it has been suggested that homocysteinylation could increase atherogenicity of LDL. Oxidative damage of endothelial cells (EC) is considered to be a critical aspect of the atherosclerotic process. To further investigate the molecular mechanisms involved in the atherogenicity of homocysteinylated LDL, we studied the effect of interaction between Hcy‐LDL and EC on cell oxidative damage, using human aortic endothelial cells (HAEC) as experimental model. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy normolipemic subjects, with HcyT (10–100 μM). In our experimental conditions, homocysteinylation treatment was not accompanied by oxidative damage of LDL. No modifications of apoprotein structure and physico‐chemical properties were observed in Hcy‐LDL with respect to control LDL (c‐LDL), as evaluated using the intrinsic fluorescence of tryptophan and the probe Laurdan incorporated in lipoproteins. Our results demonstrated that Hcy‐LDL incubated at 37°C for 3 h with HAEC, induced an oxidative damage on human EC with a significant increase of lipid hydroperoxides in cells incubated with Hcy‐LDL with respect to cell incubated with c‐LDL. The compositional changes were associated with a significant decrease viability in cells treated with Hcy‐LDL. The relationship between the levels of –SH groups of LDL and the oxidative damage of HAEC has been demonstrated. These results suggest that Hcy‐LDL exert a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of EC.

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.

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na+/K+-ATPase activity (−66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.

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.

Alzheimer's Disease and Diabetes: New Insights and Unifying Therapies

Several research groups have begun to associate the Alzheimer Disease (AD) to Diabetes Mellitus (DM), obesity and cardiovascular disease. This relationship is so close that some authors have defined Alzheimer Disease as Type 3 Diabetes. Numerous studies have shown that people with type 2 diabetes have twice the incidence of sporadic AD. Insulin deficiency or insulin resistance facilitates cerebral β-amyloidogenesis in murine model of AD, accompanied by a significant elevation in APP (Amyloid Precursor Protein) and BACE1 (β-site APP Cleaving Enzime 1). Similarly, deposits of Aβ produce a loss of neuronal surface insulin receptors and directly interfere with the insulin signaling pathway. Furthermore, as it is well known, these disorders are both associated to an increased cardiovascular risk and an altered cholesterol metabolism, so we have analyzed several therapies which recently have been suggested as a remedy to treat together AD and DM. The aim of the present review is to better understand the strengths and drawbacks of these therapies.

Oxidative stress in ischaemic stroke.

Eur J Clin Invest 2011; 41 (12): 1318–1322

Reduced susceptibility to peroxidation of erythrocyte plasma membranes from centenarians.

The plasma membrane composition affects intracellular processes and the cellular susceptibility to free radical attack, which has been associated with the impairment of cellular functions occurring during senescence. The study of the modifications of the plasma membrane in centenarians might elucidate the biological mechanisms at the basis of longevity and successful aging. The work was performed in 190 subjects, divided into five groups according to the age range: (1) 21-40 years (n=25); (2) 41-60 years (n=30); (3) 61-80 years (n=30); (4) 81-99 years (n=50); and (5) centenarians (> or = 100 years) (n=55). The following determinations were performed on erythrocyte membranes: (i) the lipid peroxide level (Lp) evaluated as malondialdehyde content; (ii) susceptibility to in vitro oxidation evaluated as difference in the content of thiobarbituric acid-reactive substances before and after phenylhydrazine addition; (iii) unsaturated/saturated fatty acid ratio and individual polyunsaturated fatty acid composition measured by gas chromatography; and (iv) fluidity studied by means of the anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Erythrocyte membranes from centenarians showed: (i) decreased basal lipid peroxide levels and reduced susceptibility to peroxidation in comparison with elderly subjects; (ii) increased unsaturated/saturated fatty acid ratio in comparison with every other age group; (iii) higher levels of eicosapentaenoic and docosahexaenoic acid and reduced content of linoleic and arachidonic acid in comparison with elderly subjects; and (iv) decreased anisotropy of TMA-DPH, i.e. higher fluidity compared with all the other age groups. In conclusion, the present work demonstrates that erythrocyte membranes from centenarians show some distinct features in comparison with elderly subjects that might act in a protective way against injuries.

Lipid peroxidation in stroke patients

Abstract Background: Paraoxonase is high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in the antioxidant and anti-inflammatory properties exerted by HDL. Increasing evidence support a role of free radicals and oxidative stress in neuronal damage induced by ischemia-reperfusion. The aim of this study was to further investigate the relationship between lipoprotein oxidative damage and stroke. Methods: We compared the paraoxonase activity and levels of lipid hydroperoxides in plasma isolated from healthy subjects (n=50) and from stroke patients (n=49). Moreover, the correlations between biochemical markers and the National Institute of Health Stroke Scale (NIHSS), which is widely used to study neurological severity, were evaluated. Results: Our results demonstrated, for the first time, that the activity of paraoxonase in plasma of stroke subjects was significantly lower than controls (p<0.001) and the levels of lipid hydroperoxides were significantly higher in plasma from patients (p<0.001). Moreover, using linear regression analysis, significant correlations between the activity of paraoxonase, lipid peroxidation and the severity of neurological deficit at admission were observed. Conclusions: These results provide further evidence that oxidative stress and impairment of the antioxidant system may play a role in stroke. Antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. Clin Chem Lab Med 2008;46:113–7.

Evidence for reduction of pro-atherosclerotic properties in platelets from healthy centenarians.

The aim of the present study was to investigate if aging is associated with platelet membrane modifications possibly related with cellular activation and hyperaggregability and if platelets from centenarians show different properties which might play a role in successful aging and longevity. Platelet plasma membranes were obtained from 60 healthy subjects, divided into four groups according to the age range: (1) 21-39 years; (2) 40-59 years; (3) 60-79 years; (4) centenarians (>/=100 years). Both centenarians and control subjects were submitted to the following inclusion criteria: liver, kidney, and thyroid function tests within the normal range; absence of history of diabetes, hypertension or coronary heart disease; no signs of edema or dehydration; no drug or vitamin supplement in the 4 weeks before the study; absence of Alzheimers disease or secondary dementia. The following determinations were performed: lipid peroxide levels (Lp) evaluated by the measurement of thiobarbituric acid (TBA) reactivity, fluidity studied by the fluorescence anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), Na(+)/K(+)-ATPase activity measured by the method of Kitao and Hattori, and sialic acid (SA) content evaluated by the periodate-thiobarbituric acid method. Centenarians showed: (i) Lp concentrations lower than elderly subjects; (ii) increased Na(+)/K(+)-ATPase activity compared with adult and elderly subjects; (iii) higher TMA-DPH anisotropy than elderly subjects; (iv) SA content similar to the young and adult groups.The present work found deep platelet membrane modifications in centenarians compared with elderly subjects. These changes are likely associated with a decreased platelet activation and therefore might exert a protective role against cardiovascular accidents, as platelet activation is a key event in the initiation and progression of arteriosclerosis.

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.