Matteo Becatti

SIRT1 modulates MAPK pathways in ischemic-reperfused cardiomyocytes.

SIRT1, an ubiquitous NAD(+)-dependent deacetylase that plays a role in biological processes such as longevity and stress response, is significantly activated in response to reactive oxygen species (ROS) production. Resveratrol (Resv), an important activator of SIRT1, has been shown to exert major health benefits in diseases associated with oxidative stress. In ischemia–reperfusion (IR) injury, a major role has been attributed to the mitogen-activated protein kinase (MAPK) pathway, which is upregulated in response to a variety of stress stimuli, including oxidative stress. In neonatal rat ventricular cardiomyocytes subjected to simulated IR, the effect of Resv-induced SIRT1 activation and the relationships with the MAPK pathway were investigated. Resv-induced SIRT1 overexpression protected cardiomyocytes from oxidative injury, mitochondrial dysfunction, and cell death induced by IR. For the first time, we demonstrate that SIRT1 overexpression positively affects the MAPK pathway—via Akt/ASK1 signaling—by reducing p38 and JNK phosphorylation and increasing ERK phosphorylation. These results reveal a new protective mechanism elicited by Resv-induced SIRT1 activation in IR tissues and suggest novel potential therapeutic targets to manage IR-induced cardiac dysfunction.

Anti-oxidant enzymes and related elements in term and preterm newborns

Background:  Although oxidative stress‐related diseases mostly affect neonates with extremely low birthweight, healthy preterm newborns might also be at risk of oxidative damages. The aim of the present study was to verify this possibility.

Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers

Increasing evidence suggests that the interaction of misfolded protein oligomers with cell membranes is a primary event resulting in the cytotoxicity associated with many protein-misfolding diseases, including neurodegenerative disorders. We describe here the results of a study on the relative contributions to toxicity of the physicochemical properties of protein oligomers and the cell membrane with which they interact. We altered the amount of cholesterol and the ganglioside GM1 in membranes of SH-SY5Y cells. We then exposed the cells to two types of oligomers of the prokaryotic protein HypF-N with different ultrastructural and cytotoxicity properties, and to oligomers formed by the amyloid-β peptide associated with Alzheimers disease. We identified that the degree of toxicity of the oligomeric species is the result of a complex interplay between the structural and physicochemical features of both the oligomers and the cell membrane.

Curcumin protects cardiac cells against ischemia-reperfusion injury: effects on oxidative stress, NF-κB, and JNK pathways

In this study we explored the effects of curcumin in cardiac cells subjected to a protocol simulating ischemia-reperfusion (IR). Curcumin (10 microM) was administered before ischemia (pretreatment) or at the moment of reperfusion (posttreatment) and its effects were compared to those produced by a reference antioxidant (Trolox) with an equal antioxidant capacity. IR cardiac cells showed clear signs of oxidative stress, impaired mitochondrial activity, and a marked development of both necrotic and apoptotic processes; at the same time, increases in NF-kappaB nuclear translocation and JNK phosphorylation were observed. Curcumin pretreatment was revealed to be the most effective in attenuating all these modifications and, in particular, in reducing the death of IR cells. This confirms that the protective effect of curcumin is not related simply to its antioxidant properties but involves other mechanisms, notably interactions in the NF-kappaB and JNK pathways. These findings suggest that curcumin administration, in particular before the hypoxic challenge, represents a promising approach to protecting cardiac cells against IR injury. In this scenario our results point out the importance of the chronology for the outcome of the treatment and provide a differential valuation of the degree of protection that curcumin can exert by its antioxidant activity or by other mechanisms.

Fibrin Resistance to Lysis in Patients with Pulmonary Hypertension Other Than Thromboembolic

RATIONALE Reportedly, fibrin isolated from patients with chronic thromboembolic pulmonary hypertension (CTEPH) is resistant to lysis. Persistence of regions within the fibrin beta chain, which mediate cell signaling and migration, could trigger the organization of pulmonary thromboemboli into chronic intravascular scars. OBJECTIVES Ascertain whether fibrin resistance to lysis occurs in patients with pulmonary hypertension (PAH) other than CTEPH, and in those with prior pulmonary embolism (PE) and no pulmonary hypertension. METHODS Fibrinogen was purified from 96 subjects (17 with CTEPH, 14 with PAH, 39 with prior PE, and 26 healthy control subjects) and exposed to thrombin to obtain fibrin clots. Plasmin-mediated cleavage of fibrin beta chain was assessed hourly over a 6-hour period by polyacrylamide gel electrophoresis. Fibrin band intensity was measured by densitometry of stained gels. Data were normalized to the band intensity of the undigested protein. MEASUREMENTS AND MAIN RESULTS By 1 hour of digestion, fibrin band intensity had decreased by a median of 25% (interquartile range [IQR], 20 to 27%) in control subjects, and by 15% (IQR, 11 to 18%) in patients with prior PE (P < 0.0001). The 1-hour median reduction in band intensity was 2% (IQR, 1 to 3%) in CTEPH, and 4% (IQR, 2 to 7%) in PAH (P < 0.0001 vs. control subjects and PE). The decline in fibrin band intensity remained significantly different among the four groups up to 6 hours (P < 0.0001). CONCLUSIONS Fibrin resistance to lysis occurs in pulmonary hypertension other than CTEPH and, to a smaller extent, in patients with prior PE and no pulmonary hypertension.

A comparison of the biochemical modifications caused by toxic and non-toxic protein oligomers in cells

Peptides and proteins can convert from their soluble forms into highly ordered fibrillar aggregates, giving rise to pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. It is increasingly recognized that protein oligomers forming early in the process of fibril aggregation represent the pathogenic species in protein deposition diseases. The N‐terminal domain of the HypF protein from Escherichia coli (HypF‐N) has previously been shown to form, under distinct conditions, two types of HypF‐N oligomers with indistinguishable morphologies but distinct structural features at the molecular level. Only the oligomer type exposing hydrophobic surfaces and possessing sufficient structural plasticity is toxic (type A), whereas the other type is benign to cultured cells (type B). Here we show that only type A oligomers are able to induce a Ca2+ influx from the cell medium to the cytosol, to penetrate the plasma membrane, to increase intracellular reactive oxygen species production, lipid peroxidation and release of intracellular calcein, resulting in the activation of the apoptotic pathway. Remarkably, these oligomers can also induce a loss of cholinergic neurons when injected into rat brains. By contrast, markers of cellular stress and viability were unaffected in cultured and rat neuronal cells exposed to type B oligomers. The analysis of the time scales of such effects indicates that the difference of toxicity between the two oligomer types involve the early events of the toxicity cascade, shedding new light on the mechanism of action of protein oligomers and on the molecular targets for the therapeutic intervention against protein deposition diseases.

The involvement of Smac/DIABLO, p53, NF-kB, and MAPK pathways in apoptosis of keratinocytes from perilesional vitiligo skin: Protective effects of curcumin and capsaicin.

Oxidative stress has been suggested as the initial pathogenetic event in melanocyte degeneration in vitiligo. Our previous results indicate that keratinocytes from perilesional skin show the features of damaged cells. In the present study, biopsies were taken from the perilesional skin of 12 patients suffering from nonsegmental vitiligo. The intracellular pathways involved in keratinocyte damage and apoptosis and the antioxidant protection of curcumin and capsaicin in these cells were investigated. In keratinocytes from perilesional vitiligo skin, we observed high levels of activated p38, NF-kB p65 subunit, p53, and Smac/DIABLO proteins. In contrast, low levels of ERK phosphorylation were present. To investigate the relationship between these pathways, we used specific inhibitors and evaluated the alteration of each pathway. For the first time, our study demonstrates the pivotal role of p38 MAP kinase as an upstream signal of perilesional keratinocyte damage, and the important contribution of p38 and NF-kB on p53 accumulation. Curcumin and capsaicin also increase ERK phosphorylation, thus inhibiting apoptosis. Moreover, pretreatment with such natural antioxidants inhibited caspase activation, increased total antioxidant capacity, repressed intracellular ROS generation and lipid peroxidation, and improved mitochondrial activity. These results suggest that antioxidants might represent an alternative approach to protect against vitiligo progression.

Role of NADPH oxidase in H9c2 cardiac muscle cells exposed to simulated ischaemia-reperfusion.

Oxidative stress is associated with several cardiovascular pathologies, including hypertension, cardiac hypertrophy and heart failure. Although oxidative stress is also increased after ischaemia‐reperfusion (I/R), little is known about the role and the activation mechanisms, in cardiac myocytes under these conditions, of NADPH oxidase, a superoxide‐producing enzyme. We found that rat cardiac muscle cells (H9c2) subjected to an in vitro simulated ischaemia (substrate‐free medium plus hypoxia) followed by ‘reperfusion’, displayed increased reactive oxygen species (ROS) production attributable to a parallel increase of NADPH oxidase activity. Our investigation on mechanisms responsible for NADPH oxidase activation showed a contribution of both the increase of NOX2 expression and p47phox translocation to the membrane. We also found that the increase of NADPH oxidase activity was associated with higher levels of lipid peroxidation, the activation of redox‐sensitive kinases, in particular ERK and JNK, and with cell death. Diphenyleneiodonium (DPI), a flavoprotein inhibitor used as NADPH oxidase inhibitor, prevented I/R‐induced ROS formation in treated cells, together with the related lipoperoxidative damage, and JNK phosphorylation without affecting ERK activation, resulting in protection against cell death. Our results provide evidence that NADPH oxidase is a key enzyme involved in I/R‐induced oxidant generation and suggest it can be a possible target in cardioprotective strategies against I/R injury, a condition of great importance in human pathology.

Protective effect of new S-acylglutathione derivatives against amyloid-induced oxidative stress

Recent data support the role of oxidative stress in the pathogenesis of Alzheimer disease (AD). In particular, glutathione (GSH) metabolism is altered and its levels are decreased in affected brain regions and peripheral cells from AD patients and in experimental models of AD. In the past decade, interest in the protective effects of various antioxidants aimed at increasing intracellular GSH content has been growing. Because much experimental evidence suggests a possible protective role of unsaturated fatty acids in age-related diseases, we designed the synthesis of new S-acylglutathione (acyl-SG) thioesters. S-Lauroylglutathione (lauroyl-SG) and S-palmitoleoylglutathione (palmitoleoyl-SG) were easily internalized into the cells and they significantly reduced Abeta42-induced oxidative stress in human neurotypic SH-SY5Y cells. In particular, acyl-SG thioesters can prevent the impairment of intracellular ROS scavengers, intracellular ROS accumulation, lipid peroxidation, and apoptotic pathway activation. Palmitoleoyl-SG seemed more effective in cellular protection against Abeta-induced oxidative damage than lauroyl-SG, suggesting a valuable role for the monounsaturated fatty acid. In this study, we demonstrate that acyl-SG derivatives completely avoid the sharp lipoperoxidation in primary fibroblasts from familial AD patients occurring after exposure to Abeta42 aggregates. Hence, we put forward these derivatives as new antioxidant compounds which could be excellent candidates for therapeutic treatment of AD and other oxidative stress-related diseases.

Oxidative Modification of Fibrinogen Is Associated With Altered Function and Structure in the Subacute Phase of Myocardial Infarction

Objective—Among plasma proteins, fibrinogen represents a major target of oxidative modifications. In patients with post–acute myocardial infarction (6 months after the acute event), fibrinogen oxidation-induced carbonyls and fibrinogen function were estimated using in vitro and ex vivo approaches. Fibrinogen structural features and clot architecture were also explored. Approach and Results—In 39 patients with post–acute myocardial infarction and 28 age-, sex-, and risk factor-matched controls, oxidative stress markers (in plasma and in purified fibrinogen fractions), thrombin-catalyzed fibrin polymerization, and plasmin-induced fibrin lysis were estimated. Circular dichroism spectra of purified fibrinogen extracts, electron microscopy, and differential interference contrast microscopy analyses of fibrin clots were also performed. Marked signs of oxidative stress in plasma (P<0.01 versus controls) and, correspondingly, an increased extent of fibrinogen carbonylation (3.5-fold over control values; P<0.01 versus controls) were observed in patients. Furthermore, fibrinogen fractions purified from patients exhibited significantly reduced clotting ability and decreased susceptibility to plasmin-induced lysis (P<0.01 versus controls). Alterations in fibrinogen secondary structure, as suggested by circular dichroism spectroscopy, and in fibrin clot architecture, as analyzed by electron and differential interference contrast microscopy, were also identified. Conclusions—Here, we report for the first time that patients with post–acute myocardial infarction present with an overall imbalance in redox status and marked fibrinogen carbonylation associated with altered fibrinogen function, thus suggesting a role for carbonylation as a direct mechanism of fibrinogen function. The observed features occur along with modifications in protein structure and in clot architecture.