Cinzia Moroni

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.

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.

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.

Peroxynitrite production and NOS expression in astrocytes U373MG incubated with lipoproteins from Alzheimer patients

Apolipoprotein E (apo E), a plasma protein involved both in the metabolism of cholesterol and triglycerides, particularly in nervous tissue, has been associated with a higher risk of Alzheimers disease. It has been shown that apo E increased the production of nitric oxide (NO) from human monocyte-derived macrophages (MDM); this effect could represent an important link between tissue redox balance and inflammation, since inflammation and oxidative stress are involved in chronic neurodegenerative disorders. Moreover, it has been evidenced that an overproduction of NO in the central nervous system (CNS) may play a key role in aging and that the glial cells (microglials cells and probably astrocytes) are able to form consistent amounts of NO through the induction of a nitric oxide synthase (iNOS) isoform so-called inducible or inflammatory. This report was performed in order to elucidate the effects produced by lipoproteins from control subjects, AD patients and first degree relatives (offspring) on human astrocyte cells after a short incubation. Peroxynitrite and NO production and NOS expression in cultured astrocytes were measured. We observed a decreased NO production after incubation with both LDL and HDL and an increased peroxynitrite production. As it concerns NOS expression, densitometric analysis of bands indicated that iNOS protein levels were significantly higher in the cells incubated with both AD lipoproteins and offspring lipoproteins compared to cells incubated with control lipoproteins. These findings suggest the possibility to identify in NO pathway a precocious marker of AD.

Alterations of platelet biochemical and functional properties in newly diagnosed type 1 diabetes: a role in cardiovascular risk?

The involvement of platelets in the pathogenesis of diabetic vascular complications is supported by several studies. Type 1 diabetic (T1D) platelets show increased adhesiveness and aggregation related to a modification of nitric oxide synthase activity. Moreover, different cell types from diabetic patients showed a decreased membrane Na+/K+‐ATPase activity, which might be involved in diabetic complications. The aim of this study was to investigate whether T1D at onset is able to induce alterations of platelet physicochemical and functional properties and whether these changes are affected by hyperglycaemia.

Homocysteinylation of low‐density lipoproteins (LDL) from subjects with Type 1 diabetes: effect on oxidative damage of human endothelial cells

Background  Homocysteine (Hcy) is an independent risk factor for cardiovascular disease (CVD). Individuals with Type 1 and Type 2 diabetes are more susceptible to the effects of homocysteine than non‐diabetic subjects. The interaction between homocysteine‐thiolactone (Hcy‐thiolactone), a reactive product of Hcy, and low‐density lipoproteins (LDL) induces the formation of homocystamide‐LDL adducts (Hcy‐LDL) and it has been suggested that homocysteinylation could increase atherogenicity of lipoproteins.

LDL and HDL affect nitric oxide metabolism in human astrocytoma cells

Astrocytes provide structural, trophic and metabolic support to neurons and modulate synaptic activity. Under physiological conditions, neuronal-derived nitric oxide (NO) plays an important role in the modulation of a variety of central nervous system (CNS) functions. NO, although short lived, can travel sufficient distances to be able to act as an intercellular messenger in the brain. Its targets include adjacent neurons and astrocytes. The aim of the present study was performed in order to investigate the effects produced by incubation of lipoproteins, at different times, with human astrocytoma cells and thus measuring NO and its metabolite production. NO and peroxynitrite production, iNOS and nNOS expression by Western immunoblot were evaluated. The LDL and HDL-treated cells showed an increased production of NO, more evident after 12 h, compared to basal levels; concerning peroxynitrite production, LDL and HDL-treated cells showed a higher fluorescence, more evident at 3 h. nNOS and iNOS protein levels were significantly higher in the cells incubated with control LDL and HDL. The present work supports the hypothesis that lipoproteins can induce the formation of reactive astrocytes, inducing iNOS as reported by other authors, giving experimental support to a role played by LDL and HDL inducing a reactive response.

Cytotoxic effects induced by hexachlorobenzene in Squilla mantis (L.) (Crustacea, Stomatopoda)

Contamination of marine environments by hexachlorobenzene (HCB) represents a serious concern for potential consequences on ecosystem and human health. Despite this, information on cytotoxic effects on marine organisms is still largely lacking. In this study, we investigated cytotoxic effects induced by HCB on gonads and muscular tissue of Squilla mantis by analysing Na+/K+‐ATPase activity and plasma membrane fluidity. This crustacean species was selected as a model for its habitat, trophic level, feeding behavior, and commercial exploitation for human consumption. Time course experiments revealed that low concentrations of HCB (i.e. 50 nM) determine an exponentially decrease of Na+/K+‐ATPase activity and a significant modification of cellular membrane fluidity. Significant negative relationships between Na+/K+‐ATPase activity and membrane fluidity were observed, suggesting that changes in the structure and packing of cellular membranes induced by HCB may be the primary factor affecting the activity of essential bilayer‐associated enzymes. Overall these findings suggest that even small concentrations of HCB may determine important changes on cell metabolism with potential cascade effects on recruitment of this commercial species.