Paolo Nassi

Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer’s patients

We have measured the levels of typical end products of the processes of lipid peroxidation, protein oxidation, and total antioxidant capacity (TAC) in skin fibroblasts and lymphoblasts taken from patients with familial Alzheimers disease (FAD), sporadic Alzheimers disease (AD), and age-matched healthy controls. Compared to controls, the fibroblasts and lymphoblasts carrying amyloid precursor protein (APP) and presenilin-1 (PS-1) gene mutations showed a clear increase in lipoperoxidation products, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In contrast, the antioxidant defenses of cells from FAD patients were lower than those from normal subjects. Lipoperoxidation and antioxidant capacity in lymphoblasts from patients affected by sporadic AD were virtually indistinguishable from the basal values of normal controls. An oxidative attack on protein gave rise to greater protein carbonyl content in FAD patients than in age-matched controls. Furthermore, ADP ribosylation levels of poly(ADP-ribose) polymerase (PARP) nuclear substrates were significantly raised, whereas the PARP content did not differ significantly between fibroblasts carrying gene mutations and control cells. These results indicate that peripheral cells carrying APP and PS-1 gene mutations show altered levels of oxidative markers even though they are not directly involved in the neurodegenerative process of AD. These results support the hypothesis that oxidative damage to lipid, protein, and DNA is an important early event in the pathogenesis of AD.

Changes in Na+,K-ATPase, Ca2-ATPase and some soluble enzymes related to energy metabolism in brains of patients with Alzheimer's disease

Hexokinase, lactate dehydrogenase, acylphosphatase, (Na+,K+)-ATPase and Ca2(+)-ATPase of selected areas from postmortem Alzheimers disease brains were studied. Hexokinase and lactate dehydrogenase were significantly changed in all the examined subcortical nuclei. (Na+,K+)-ATPase activity was altered in several areas of Alzheimers disease brains. No changes in Ca2(+)-ATPase and acylphosphatase were observed. The main alterations of the assayed enzymes were observed in subcortical areas but not in cortical areas of Alzheimers disease brains.

Oxidative stress and antioxidant defenses in renal patients receiving regular haemodialysis

Abstract Patients with chronic renal failure, and particularly those receiving regular haemodialysis, have a high incidence of premature cardiovascular disease. Oxidative stress, which causes lipid peroxidation, may contribute to increase the risk of atherosclerosis. The results of the present study indicate that lipid peroxidation products (malonaldehyde and 4-hydroxyalkenals) are significantly increased in plasma of renal patients before dialysis and, although reduced, remained above the normal range after this treatment. Moreover, production of free radicals and reactive oxygen metabolites was increased in chronic renal failure patients, especially after dialysis. On the other hand, the antioxidant defenses of those patients were higher than those of normal subjects, as judged from the plasma levels of specific antioxidant molecules and from the plasma antioxidant capacity. We also found that triglycerides were significantly higher in renal patients, both before and after dialysis, than in the control group. These results suggest that patients on chronic haemodialysis are particularly prone to oxidative stress and that dialysis itself may worsen this condition. Rather than to a weakening of antioxidant defenses, the susceptibility of chronic renal failure patients to oxidative stress might be ascribed to an increased free radical and reactive oxygen metabolite production and to increased levels of oxidizable substrates, notably triglycerides with their unsaturated fatty acids.

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.

Enhanced ROS production by NADPH oxidase is correlated to changes in antioxidant enzyme activity in human heart failure

In pathological conditions, the balance between reactive oxygen species (ROS) and antioxidants may shift toward a relative increase of ROS, resulting in oxidative stress. Conflicting data are available on antioxidant defenses in human failing heart and they are limited to the left ventricle. Thus, we aimed to investigate and compare the source of oxidant and antioxidant enzyme activities in the right (RV) and left (LV) ventricles of human failing hearts. We found a significant increase in superoxide production only by NADPH oxidase in both failing ventricles, more marked in RV. Despite unchanged mRNA or protein expression, catalase (CAT) and glutathione peroxidase (GPx) activities were increased, and their increases reflected the levels of Tyr phosphorylation of the respective enzyme. Manganese superoxide dismutase (Mn-SOD) activity appeared unchanged. The increase in NADPH oxidase-dependent superoxide production positively correlated with the activation of both CAT and GPx. However, the slope of the linear correlation (m) was steeper in LV than in RV for GPx (LV: m=2.416; RV: m=1.485) and CAT (LV: m=1.007; RV: m=0.354). Accordingly, malondialdehyde levels, an indirect index of oxidative stress, were significantly higher in the RV than LV. We conclude that in human failing RV and LV, oxidative stress is associated with activation of antioxidant enzyme activity. This activation is likely due to post-translational modifications and more evident in LV. Overall, these findings suggest a reduced protection of RV against oxidative stress and its potential contribution to the progression toward overt heart failure.

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.

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 effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts

Abstract.To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD+ and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD+ and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion.