In the present study we tested the effect of small molecular weight redox molecules on collagen-i... more In the present study we tested the effect of small molecular weight redox molecules on collagen-induced platelet aggregation. We used N-acetylcysteine amide (AD4), the amide form of N-acetylcysteine (NAC), a thiol antioxidant with improved lipophilicity and bio-availability compared to NAC, and the thioredoxin mimetics (TXM) peptides, TXM-CB3, TXM-CB13, and TXM-CB30. All compounds significantly inhibited platelet aggregation induced by collagen, with TXM-peptides and AD4 being more effective than NAC. The levels of TxB2 and 12-HETE, the main metabolites derived from the cyclooxygenase and lipoxygenase pathways following platelet activation, were significantly reduced in the presence of AD4, TXM-peptides, or NAC, when tested at the highest concentration (0.6 mM). The effect of AD4, TXM-peptides, and NAC was tested also on the clotting time (CT) of whole blood. TXM-CB3 and TXM-CB30 showed the highest increase of CT. Furthermore, two representative compounds, TXM-CB3, and NAC, showed a...
In the present study, we tested the effect of small-molecular-weight redox molecules on collagen-... more In the present study, we tested the effect of small-molecular-weight redox molecules on collagen-induced platelet aggregation. We used N-acetylcysteine amide (AD4/NACA), the amide form of N-acetylcysteine (NAC), a thiol antioxidant with improved lipophilicity and bioavailability compared to NAC, and the thioredoxin-mimetic (TXM) peptides, TXM-CB3, TXM-CB13, and TXM-CB30. All compounds significantly inhibited platelet aggregation induced by collagen, with TXM-peptides and AD4 being more effective than NAC. The levels of TxB2 and 12-HETE, the main metabolites derived from the cyclooxygenase and lipoxygenase pathways following platelet activation, were significantly reduced in the presence of AD4, TXM peptides, or NAC, when tested at the highest concentration (0.6 mM). The effects of AD4, TXM-peptides, and NAC were also tested on the clotting time (CT) of whole blood. TXM-CB3 and TXM-CB30 showed the greatest increase in CT. Furthermore, two representative compounds, TXM-CB3 and NAC, sh...
The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes.... more The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes. Excessive adipogenesis promotes obesity, a condition that increasingly threatens global health and contributes to the rapid rise of obesity-related diseases. We have recently shown that prenylcysteine oxidase 1 (PCYOX1) is a regulator of atherosclerosis-disease mechanisms, which acts through mechanisms not exclusively related to its pro-oxidant activity. To address the role of PCYOX1 in the adipogenic process, we extended our previous observations confirming that Pcyox1−/−/Apoe−/− mice fed a high-fat diet for 8 or 12 weeks showed significantly lower body weight, when compared to Pcyox1+/+/Apoe−/− mice, due to an evident reduction in visceral adipose content. We herein assessed the role of PCYOX1 in adipogenesis. Here, we found that PCYOX1 is expressed in adipose tissue, and, independently from its pro-oxidant enzymatic activity, is critical for adipogenesis. Pcyox1 gene silencing comple...
Macrophages are heterogeneous and plastic cells, able to adapt their phenotype and functions to c... more Macrophages are heterogeneous and plastic cells, able to adapt their phenotype and functions to changes in the microenvironment. They are involved in several homeostatic processes and also in many human diseases, including atherosclerosis, where they participate in all the stages of the disease. For these reasons, macrophages have been studied extensively using different approaches, including proteomics. Proteomics, indeed, may be a powerful tool to better understand the behavior of these cells, and a careful analysis of the proteome of different macrophage phenotypes can help to better characterize the role of these phenotypes in atherosclerosis and provide a broad view of proteins that might potentially affect the course of the disease. In this review, we discuss the different proteomic techniques that have been used to delineate the proteomic profile of macrophage phenotypes and summarize some results that can help to elucidate the roles of macrophages and develop new strategies ...
Experimental studies suggest that red blood cells are involved in nitric oxide (NO) synthesis and... more Experimental studies suggest that red blood cells are involved in nitric oxide (NO) synthesis and delivery. Erythrocytes acts as producers, scavengers and vehicles of NO affecting several physiological processes. NO bioavailability is linked not only to arginine and its metabolic products, ornithine and citrulline, but also to methylarginines which are inhibitors of NO sinthesis. Existing methodologies do not permit a systematic evaluation of the metabolic and biosynthetic pathway of NO in RBC. A metabolomic approach, considering a larger number of compounds involved in NO metabolic pathway, could be important to better understand the role of red blood cells in physiological and pathological conditions. We set up an HPLC-electrospray ionization-tandem mass spectrometry method, to simultaneously detect and quantify arginine metabolome, i.e. arginine, symmetric and asymmetric dimethylarginine, monomethyarginine, ornithine, citrulline. Stable isotope-labelled internal standards have been used to minimize analytical variations before organic protein precipitation of the isolated and lysated erythrocytes. The ion source was operating in positive ion mode and the analytes were detected using multiple reaction monitoring after hydrophilic-interaction chromatography. A 8 min run time allowed the simultaneous evaluation of all the analytes involved in the NO pathway. The chromatograms were free from interferences, the calibration functions were linear and the inter-day CV were<10% for all the analytes. LOQ values make this method suitable for low concentration samples. Our validated LC-MS/MS method might be useful to clarify the role of RBCs in the synthesis of NO and will be of help for studies assessing the involvement of these cells in the regulation of blood flow under physiologic or pathological conditions. Moreover, a simple sample processing without the derivatization step, allows to consider this method a valuable tool for diagnostic evaluation of arginine metabolome in red blood cells and it could be applied in clinical chemistry
In recent years the role of lipids and/or lipoproteins in the occurrence and development of ather... more In recent years the role of lipids and/or lipoproteins in the occurrence and development of atherosclerotic lesions has been elucidated. On the other hand low density lipoproteins (LDL) have been recognized to interfere with functional and biochemical aspects not only of the vascular wall, i. e. endothelial cells and smooth muscle cells, but also of blood cells involved in the thrombotic processes. The present knowledge on the interaction between atherogenic lipoproteins and cells involved in the thrombotic processes is discussed.
Biochimica et Biophysica Acta - Lipids and Lipid Metabolism, 1993
Eight normal volunteers (four men and four women) were treated with 3 x 1 g capsules of n-3 fatty... more Eight normal volunteers (four men and four women) were treated with 3 x 1 g capsules of n-3 fatty acid ethyl esters for a period of 18 weeks, followed by a 24 week washout. Fatty acids of plasma, platelets, monocytes and red blood cells were analyzed at 0, 6, 12 and 18 weeks of treatment and at 4, 14 and 24 weeks of washout. During treatment, accumulation of EPA in plasma and cells was almost maximal at 6 weeks, whereas that of DHA reached a peak at 18 weeks. Arachidonic acid declined somewhat at 12 weeks in plasma and more markedly at 18 weeks in red blood cells and monocytes. During washout, EPA returned rapidly toward pretreatment values in all compartments, but it remained significantly higher in plasma and platelets at the end of washout. DHA declined more slowly, maintaining higher than basal values in plasma and platelets and lower than basal in red blood cells, at the end of washout. Rebound increments of AA occurred in plasma. Finally, the plasma levels of AA, but not those of the n-3 fatty acids, were more markedly modified in males than in females. The presented results suggest interactions between circulating fatty acids in the different compartment after n-3 FA administration, and indicate that very long washouts are necessary for a complete recovery from the induced fatty acid modifications.
b ´´ ´ Abstract Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppres... more b ´´ ´ Abstract Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages.
Early post-operative aspirin improves survival in patients undergoing coronary artery bypass graf... more Early post-operative aspirin improves survival in patients undergoing coronary artery bypass graft (CABG). However, most patients do not benefit of aspirin after CABG, still remaining at risk of thrombotic events due to insufficient platelet inhibition, specifically via the thromboxane (TX) pathway. We evaluated the effect of two aspirin doses (100 or 325 mg daily, enteric coated formulations) on platelet function and TX biosynthesis in patients after CABG and assessed whether the incidence of residual platelet reactivity could be reduced by the higher dose. Fifty-six patients undergoing CABG were randomly assigned to 100 or 325 mg aspirin daily for five days in a prospective single-centre study. Treatment effect was assessed by measuring either platelet function (light-transmission aggregometry and point-of-care PFA-100(R)) or TX biosynthesis in collagen-stimulated platelets, serum, urine, and in lipopolysaccharide (LPS)-cultured whole blood (WB). An insufficient TX inhibition was observed with 100 mg aspirin but not with the higher dose. The different effect of the two doses was, however, highlighted by either TX (platelet- or serum-derived) or by PFA-100(R) but not by the other assays. In conclusion, early after CABG, the incidence of residual platelet activity was lower in patients who received 325 mg aspirin. Moreover, evidence was provided that different methods yield different results in the detection of aspirin resistance, rendering them not interchangeable.
Prostaglandins, Leukotrienes and Essential Fatty Acids, 2007
The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of ... more The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of distinct processes: intake, metabolism and peripheral utilization. was to compare the FA profile in plasma, lipoproteins and blood cells with that in whole blood (WB) from healthy volunteers; to assess the quantitative distribution of selected FA in triacylglycerols, cholesteryl esters and phospholipids. Lipid FA profiles are comparable in plasma and lipoproteins but differ from those in blood cells. In WB, the FA profile results from the balanced proportion of FA pools in plasma and cells. The contribution of each lipid class to the total amount of FA differs among blood specimens. Phospholipids of plasma and red blood cell are the major contributors to the FA amount and profile in WB. In conclusion, the FA profile of WB reflects the FA status and WB could be an adequate specimen for the assessment of FA intakes.
In the present study we tested the effect of small molecular weight redox molecules on collagen-i... more In the present study we tested the effect of small molecular weight redox molecules on collagen-induced platelet aggregation. We used N-acetylcysteine amide (AD4), the amide form of N-acetylcysteine (NAC), a thiol antioxidant with improved lipophilicity and bio-availability compared to NAC, and the thioredoxin mimetics (TXM) peptides, TXM-CB3, TXM-CB13, and TXM-CB30. All compounds significantly inhibited platelet aggregation induced by collagen, with TXM-peptides and AD4 being more effective than NAC. The levels of TxB2 and 12-HETE, the main metabolites derived from the cyclooxygenase and lipoxygenase pathways following platelet activation, were significantly reduced in the presence of AD4, TXM-peptides, or NAC, when tested at the highest concentration (0.6 mM). The effect of AD4, TXM-peptides, and NAC was tested also on the clotting time (CT) of whole blood. TXM-CB3 and TXM-CB30 showed the highest increase of CT. Furthermore, two representative compounds, TXM-CB3, and NAC, showed a...
In the present study, we tested the effect of small-molecular-weight redox molecules on collagen-... more In the present study, we tested the effect of small-molecular-weight redox molecules on collagen-induced platelet aggregation. We used N-acetylcysteine amide (AD4/NACA), the amide form of N-acetylcysteine (NAC), a thiol antioxidant with improved lipophilicity and bioavailability compared to NAC, and the thioredoxin-mimetic (TXM) peptides, TXM-CB3, TXM-CB13, and TXM-CB30. All compounds significantly inhibited platelet aggregation induced by collagen, with TXM-peptides and AD4 being more effective than NAC. The levels of TxB2 and 12-HETE, the main metabolites derived from the cyclooxygenase and lipoxygenase pathways following platelet activation, were significantly reduced in the presence of AD4, TXM peptides, or NAC, when tested at the highest concentration (0.6 mM). The effects of AD4, TXM-peptides, and NAC were also tested on the clotting time (CT) of whole blood. TXM-CB3 and TXM-CB30 showed the greatest increase in CT. Furthermore, two representative compounds, TXM-CB3 and NAC, sh...
The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes.... more The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes. Excessive adipogenesis promotes obesity, a condition that increasingly threatens global health and contributes to the rapid rise of obesity-related diseases. We have recently shown that prenylcysteine oxidase 1 (PCYOX1) is a regulator of atherosclerosis-disease mechanisms, which acts through mechanisms not exclusively related to its pro-oxidant activity. To address the role of PCYOX1 in the adipogenic process, we extended our previous observations confirming that Pcyox1−/−/Apoe−/− mice fed a high-fat diet for 8 or 12 weeks showed significantly lower body weight, when compared to Pcyox1+/+/Apoe−/− mice, due to an evident reduction in visceral adipose content. We herein assessed the role of PCYOX1 in adipogenesis. Here, we found that PCYOX1 is expressed in adipose tissue, and, independently from its pro-oxidant enzymatic activity, is critical for adipogenesis. Pcyox1 gene silencing comple...
Macrophages are heterogeneous and plastic cells, able to adapt their phenotype and functions to c... more Macrophages are heterogeneous and plastic cells, able to adapt their phenotype and functions to changes in the microenvironment. They are involved in several homeostatic processes and also in many human diseases, including atherosclerosis, where they participate in all the stages of the disease. For these reasons, macrophages have been studied extensively using different approaches, including proteomics. Proteomics, indeed, may be a powerful tool to better understand the behavior of these cells, and a careful analysis of the proteome of different macrophage phenotypes can help to better characterize the role of these phenotypes in atherosclerosis and provide a broad view of proteins that might potentially affect the course of the disease. In this review, we discuss the different proteomic techniques that have been used to delineate the proteomic profile of macrophage phenotypes and summarize some results that can help to elucidate the roles of macrophages and develop new strategies ...
Experimental studies suggest that red blood cells are involved in nitric oxide (NO) synthesis and... more Experimental studies suggest that red blood cells are involved in nitric oxide (NO) synthesis and delivery. Erythrocytes acts as producers, scavengers and vehicles of NO affecting several physiological processes. NO bioavailability is linked not only to arginine and its metabolic products, ornithine and citrulline, but also to methylarginines which are inhibitors of NO sinthesis. Existing methodologies do not permit a systematic evaluation of the metabolic and biosynthetic pathway of NO in RBC. A metabolomic approach, considering a larger number of compounds involved in NO metabolic pathway, could be important to better understand the role of red blood cells in physiological and pathological conditions. We set up an HPLC-electrospray ionization-tandem mass spectrometry method, to simultaneously detect and quantify arginine metabolome, i.e. arginine, symmetric and asymmetric dimethylarginine, monomethyarginine, ornithine, citrulline. Stable isotope-labelled internal standards have been used to minimize analytical variations before organic protein precipitation of the isolated and lysated erythrocytes. The ion source was operating in positive ion mode and the analytes were detected using multiple reaction monitoring after hydrophilic-interaction chromatography. A 8 min run time allowed the simultaneous evaluation of all the analytes involved in the NO pathway. The chromatograms were free from interferences, the calibration functions were linear and the inter-day CV were<10% for all the analytes. LOQ values make this method suitable for low concentration samples. Our validated LC-MS/MS method might be useful to clarify the role of RBCs in the synthesis of NO and will be of help for studies assessing the involvement of these cells in the regulation of blood flow under physiologic or pathological conditions. Moreover, a simple sample processing without the derivatization step, allows to consider this method a valuable tool for diagnostic evaluation of arginine metabolome in red blood cells and it could be applied in clinical chemistry
In recent years the role of lipids and/or lipoproteins in the occurrence and development of ather... more In recent years the role of lipids and/or lipoproteins in the occurrence and development of atherosclerotic lesions has been elucidated. On the other hand low density lipoproteins (LDL) have been recognized to interfere with functional and biochemical aspects not only of the vascular wall, i. e. endothelial cells and smooth muscle cells, but also of blood cells involved in the thrombotic processes. The present knowledge on the interaction between atherogenic lipoproteins and cells involved in the thrombotic processes is discussed.
Biochimica et Biophysica Acta - Lipids and Lipid Metabolism, 1993
Eight normal volunteers (four men and four women) were treated with 3 x 1 g capsules of n-3 fatty... more Eight normal volunteers (four men and four women) were treated with 3 x 1 g capsules of n-3 fatty acid ethyl esters for a period of 18 weeks, followed by a 24 week washout. Fatty acids of plasma, platelets, monocytes and red blood cells were analyzed at 0, 6, 12 and 18 weeks of treatment and at 4, 14 and 24 weeks of washout. During treatment, accumulation of EPA in plasma and cells was almost maximal at 6 weeks, whereas that of DHA reached a peak at 18 weeks. Arachidonic acid declined somewhat at 12 weeks in plasma and more markedly at 18 weeks in red blood cells and monocytes. During washout, EPA returned rapidly toward pretreatment values in all compartments, but it remained significantly higher in plasma and platelets at the end of washout. DHA declined more slowly, maintaining higher than basal values in plasma and platelets and lower than basal in red blood cells, at the end of washout. Rebound increments of AA occurred in plasma. Finally, the plasma levels of AA, but not those of the n-3 fatty acids, were more markedly modified in males than in females. The presented results suggest interactions between circulating fatty acids in the different compartment after n-3 FA administration, and indicate that very long washouts are necessary for a complete recovery from the induced fatty acid modifications.
b ´´ ´ Abstract Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppres... more b ´´ ´ Abstract Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages.
Early post-operative aspirin improves survival in patients undergoing coronary artery bypass graf... more Early post-operative aspirin improves survival in patients undergoing coronary artery bypass graft (CABG). However, most patients do not benefit of aspirin after CABG, still remaining at risk of thrombotic events due to insufficient platelet inhibition, specifically via the thromboxane (TX) pathway. We evaluated the effect of two aspirin doses (100 or 325 mg daily, enteric coated formulations) on platelet function and TX biosynthesis in patients after CABG and assessed whether the incidence of residual platelet reactivity could be reduced by the higher dose. Fifty-six patients undergoing CABG were randomly assigned to 100 or 325 mg aspirin daily for five days in a prospective single-centre study. Treatment effect was assessed by measuring either platelet function (light-transmission aggregometry and point-of-care PFA-100(R)) or TX biosynthesis in collagen-stimulated platelets, serum, urine, and in lipopolysaccharide (LPS)-cultured whole blood (WB). An insufficient TX inhibition was observed with 100 mg aspirin but not with the higher dose. The different effect of the two doses was, however, highlighted by either TX (platelet- or serum-derived) or by PFA-100(R) but not by the other assays. In conclusion, early after CABG, the incidence of residual platelet activity was lower in patients who received 325 mg aspirin. Moreover, evidence was provided that different methods yield different results in the detection of aspirin resistance, rendering them not interchangeable.
Prostaglandins, Leukotrienes and Essential Fatty Acids, 2007
The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of ... more The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of distinct processes: intake, metabolism and peripheral utilization. was to compare the FA profile in plasma, lipoproteins and blood cells with that in whole blood (WB) from healthy volunteers; to assess the quantitative distribution of selected FA in triacylglycerols, cholesteryl esters and phospholipids. Lipid FA profiles are comparable in plasma and lipoproteins but differ from those in blood cells. In WB, the FA profile results from the balanced proportion of FA pools in plasma and cells. The contribution of each lipid class to the total amount of FA differs among blood specimens. Phospholipids of plasma and red blood cell are the major contributors to the FA amount and profile in WB. In conclusion, the FA profile of WB reflects the FA status and WB could be an adequate specimen for the assessment of FA intakes.
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