Timoteo Marchini

Skin Damage Mechanisms Related to Airborne Particulate Matter Exposure.

Epidemiological studies suggest a correlation between increased airborne particulate matter (PM) and adverse health effects. The mechanisms of PM-health effects are believed to involve oxidative stress and inflammation. To evaluate the ability of PM promoting skin tissue damage, one of the main organs exposed to outdoor pollutants, we analyzed the effect of concentrated ambient particles (CAPs) in a reconstructed human epidermis (RHE) model. RHE tissues were exposed to 25 or 100 µg/ml CAPs for 24 or 48 h. Data showed that RHE seems to be more susceptible to CAPs-induced toxicity after 48 h exposure than after 24 h. We found a local reactive O(2) species (ROS) production increase generated from metals present on the particle, which contributes to lipids oxidation. Furthermore, as a consequence of altered redox status, NFkB nucleus translocation was increase upon CAPs exposure, as well as cyclooxygenase 2 and cytochrome P450 levels, which may be involved in the inflammatory response initiated by PM. CAPs also triggered an apoptotic process in skin. Surprisingly, by transition electron microscopy analysis we showed that CAPs were able to penetrate skin tissues. These findings contribute to the understanding of the cutaneous pathophysiological mechanisms initiated by CAPs exposure, where oxidative stress and inflammation may play predominant roles.

Lung oxidative metabolism after exposure to ambient particles

The aim of this work was to study the time course of the oxidative metabolism in mice lung after exposure to ambient particles (ROFA). Swiss mice were intranasally instilled with a ROFA suspension (0.20 mg/kg). Animals were sacrificed 1 or 3 h after the exposure. Eighty percentage of increased oxygen consumption was observed in tissue cubes after 1 h of exposure. This observation was accompanied by an increased NADPH oxidase activity (40%) and mitochondrial oxygen consumption in state 3 (19%). NO production by lung homogenates was found to be increased by 43% after 3 h of exposure. Phospholipid oxidation in lung homogenates showed a 29% increase after 1 h of exposure, while a 30% increase in the carbonyl content was found only after 3 h of exposure. Our data show the relative importance of different sources of reactive oxygen species (NADPH oxidase activity and mitochondrial respiration) to the increased tissue oxygen consumption, oxidative damage and antioxidant status observed in an acute model of ROFA particles exposure.

Time course of systemic oxidative stress and inflammatory response induced by an acute exposure to Residual Oil Fly Ash

It is suggested that systemic oxidative stress and inflammation play a central role in the onset and progression of cardiovascular diseases associated with the exposure to particulate matter (PM). The aim of this work was to evaluate the time changes of systemic markers of oxidative stress and inflammation, after an acute exposure to Residual Oil Fly Ash (ROFA). Female Swiss mice were intranasally instilled with a ROFA suspension (1.0mg/kg body weight) or saline solution, and plasma levels of oxidative damage markers [thiobarbituric acid reactive substances (TBARSs) and protein carbonyls], antioxidant status [reduced (GSH) and oxidized (GSSG) glutathione, ascorbic acid levels, and superoxide dismutase (SOD) activity], cytokines levels, and intravascular leukocyte activation were evaluated after 1, 3 or 5h of exposure. Oxidative damage to lipids and decreased GSH/GSSG ratio were observed in ROFA-exposed mice as early as 1h. Afterwards, increased protein oxidation, decreased ascorbic acid content and SOD activity were found in this group at 3h. The onset of an adaptive response was observed at 5h after the ROFA exposure, as indicated by decreased TBARS plasma content and increased SOD activity. The observed increase in oxidative damage to plasma macromolecules, together with systemic antioxidants depletion, may be a consequence of a systemic inflammatory response triggered by the ROFA exposure, since increased TNF-α and IL-6 plasma levels and polymorphonuclear leukocytes activation was found at every evaluated time point. These findings contribute to the understanding of the increase in cardiovascular morbidity and mortality, in association with environmental PM inhalation.

Reactive oxygen species produced by NADPH oxidase and mitochondrial dysfunction in lung after an acute exposure to Residual Oil Fly Ashes

Reactive O2 species production triggered by particulate matter (PM) exposure is able to initiate oxidative damage mechanisms, which are postulated as responsible for increased morbidity along with the aggravation of respiratory diseases. The aim of this work was to quantitatively analyse the major sources of reactive O2 species involved in lung O2 metabolism after an acute exposure to Residual Oil Fly Ashes (ROFAs). Mice were intranasally instilled with a ROFA suspension (1.0mg/kg body weight), and lung samples were analysed 1h after instillation. Tissue O2 consumption and NADPH oxidase (Nox) activity were evaluated in tissue homogenates. Mitochondrial respiration, respiratory chain complexes activity, H2O2 and ATP production rates, mitochondrial membrane potential and oxidative damage markers were assessed in isolated mitochondria. ROFA exposure was found to be associated with 61% increased tissue O2 consumption, a 30% increase in Nox activity, a 33% increased state 3 mitochondrial O2 consumption and a mitochondrial complex II activity increased by 25%. During mitochondrial active respiration, mitochondrial depolarization and a 53% decreased ATP production rate were observed. Neither changes in H2O2 production rate, nor oxidative damage in isolated mitochondria were observed after the instillation. After an acute ROFA exposure, increased tissue O2 consumption may account for an augmented Nox activity, causing an increased O2(-) production. The mitochondrial function modifications found may prevent oxidative damage within the organelle. These findings provide new insights to the understanding of the mechanisms involving reactive O2 species production in the lung triggered by ROFA exposure.

Cardiac mitochondrial biogenesis in endotoxemia is not accompanied by mitochondrial function recovery

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0-24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.

Myocardial triggers involved in remote ischemic preconditioning activation

New Findings What is the central question of this study? Ischemia/reperfusion of peripheral tissues protects the heart from subsequent myocardial ischemia/reperfusion injury, a phenomenon referred to as remote ischemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? rIPC reduces infarct size through a vagal pathway and a mechanism involving Akt and eNOS phosphorylation, opening of mitochondrial K+ATP channels and increasing of mitochondrial H2O2 production. All these phenomena occur prior to the myocardial ischemia, therefore they could act as “triggers” of rIPC. Aims: It has been proposed that remote ischemic preconditioning (rIPC) activates a parasympathetic neural pathway. However, the myocardial rIPC intracellular mechanism remains unclear. Here we characterized some of the intracellular signals participating as rIPC triggers. Methods and Results: Isolated rat hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion (Non-rIPC). In a second group, before the isolation of the heart, a rIPC protocol (3 cycles of hindlimb ischemia/reperfusion) was performed. The infarct size was measured with tetrazolium staining. Akt and eNOS expression/phosphorylation, and mitochondrial H2O2 production were evaluated at the end of rIPC protocol, before myocardial ischemia/reperfusion. rIPC significantly decreased the infarct size and induced Akt and eNOS phosphorylation. The protective effect on infarct size was abolished by cervical vagal section (CVS), L-NAME (NO synthesis inhibitor) and 5-HD (mK+ATP channels blocker). Mitochondrial production of H2O2 was increased by rIPC while it was abolished by CVS, L-NAME and 5-HD. Conclusions: rIPC activates a parasympathetic vagal pathway and a mechanism involving the Akt and eNOS phosphorylation, the opening of mK+ATP, and the release of H2O2 by the mitochondria. These entire phenomena occur prior to the myocardial ischemia, which could act as triggers of rIPC. This article is protected by copyright. All rights reservedWhat is the central question of this study? Ischaemia–reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia–reperfusion injury, a phenomenon referred to as remote ischaemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? Remote ischaemic preconditioning reduces infarct size through a vagal pathway and a mechanism involving phosphorylation of Akt and endothelial nitric oxide synthase, opening of mitochondrial ATP‐dependent K+ channels and an increase in mitochondrial H2O2 production. All these phenomena occur before the myocardial ischaemia; hence, they could act as ‘triggers’ of rIPC.

Myocardial triggers involved in activation of remote ischaemic preconditioning.

New Findings What is the central question of this study? Ischemia/reperfusion of peripheral tissues protects the heart from subsequent myocardial ischemia/reperfusion injury, a phenomenon referred to as remote ischemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? rIPC reduces infarct size through a vagal pathway and a mechanism involving Akt and eNOS phosphorylation, opening of mitochondrial K+ATP channels and increasing of mitochondrial H2O2 production. All these phenomena occur prior to the myocardial ischemia, therefore they could act as “triggers” of rIPC. Aims: It has been proposed that remote ischemic preconditioning (rIPC) activates a parasympathetic neural pathway. However, the myocardial rIPC intracellular mechanism remains unclear. Here we characterized some of the intracellular signals participating as rIPC triggers. Methods and Results: Isolated rat hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion (Non-rIPC). In a second group, before the isolation of the heart, a rIPC protocol (3 cycles of hindlimb ischemia/reperfusion) was performed. The infarct size was measured with tetrazolium staining. Akt and eNOS expression/phosphorylation, and mitochondrial H2O2 production were evaluated at the end of rIPC protocol, before myocardial ischemia/reperfusion. rIPC significantly decreased the infarct size and induced Akt and eNOS phosphorylation. The protective effect on infarct size was abolished by cervical vagal section (CVS), L-NAME (NO synthesis inhibitor) and 5-HD (mK+ATP channels blocker). Mitochondrial production of H2O2 was increased by rIPC while it was abolished by CVS, L-NAME and 5-HD. Conclusions: rIPC activates a parasympathetic vagal pathway and a mechanism involving the Akt and eNOS phosphorylation, the opening of mK+ATP, and the release of H2O2 by the mitochondria. These entire phenomena occur prior to the myocardial ischemia, which could act as triggers of rIPC. This article is protected by copyright. All rights reservedWhat is the central question of this study? Ischaemia–reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia–reperfusion injury, a phenomenon referred to as remote ischaemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? Remote ischaemic preconditioning reduces infarct size through a vagal pathway and a mechanism involving phosphorylation of Akt and endothelial nitric oxide synthase, opening of mitochondrial ATP‐dependent K+ channels and an increase in mitochondrial H2O2 production. All these phenomena occur before the myocardial ischaemia; hence, they could act as ‘triggers’ of rIPC.

Selective TNF-α targeting with infliximab attenuates impaired oxygen metabolism and contractile function induced by an acute exposure to air particulate matter

Inflammation plays a central role in the onset and progression of cardiovascular diseases associated with the exposure to air pollution particulate matter (PM). The aim of this work was to analyze the cardioprotective effect of selective TNF-α targeting with a blocking anti-TNF-α antibody (infliximab) in an in vivo mice model of acute exposure to residual oil fly ash (ROFA). Female Swiss mice received an intraperitoneal injection of infliximab (10 mg/kg body wt) or saline solution, and were intranasally instilled with a ROFA suspension (1 mg/kg body wt). Control animals were instilled with saline solution and handled in parallel. After 3 h, heart O2 consumption was assessed by high-resolution respirometry in left ventricle tissue cubes and isolated mitochondria, and ventricular contractile reserve and lusitropic reserve were evaluated according to the Langendorff technique. ROFA instillation induced a significant decrease in tissue O2 consumption and active mitochondrial respiration by 32 and 31%, respectively, compared with the control group. While ventricular contractile state and isovolumic relaxation were not altered in ROFA-exposed mice, impaired contractile reserve and lusitropic reserve were observed in this group. Infliximab pretreatment significantly attenuated the decrease in heart O2 consumption and prevented the decrease in ventricular contractile and lusitropic reserve in ROFA-exposed mice. Moreover, infliximab-pretreated ROFA-exposed mice showed conserved left ventricular developed pressure and cardiac O2 consumption in response to a β-adrenergic stimulus with isoproterenol. These results provides direct evidence linking systemic inflammation and altered cardiac function following an acute exposure to PM and contribute to the understanding of PM-associated cardiovascular morbidity and mortality.

Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning.

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30min of ischemia and 120min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6±2.4 vs. 39.2±2.1%, p<0.05), but this protection was abolished in DN-Trx1-PostC group (49.7±1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9±2.1 and 38.4±1.3%, p<0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3β phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3β survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.

Role of transition metals present in air particulate matter on lung oxygen metabolism.

Several epidemiological studies have shown a positive correlation between daily increases in airborne particulate matter (PM) concentration and the occurrence of respiratory and cardiovascular diseases. Transition metals present in air PM were associated with adverse health effects after PM exposure. The aim of this work was to study lung O2 metabolism after an acute exposure to transition metal-coated nanoparticles (NPs). Female Swiss mice (25g) were intranasally instilled with a suspension of silica NP containing Ni (II), Cd (II), Fe (III), or Cr (VI) at 0, 0.01, 0.05, 0.1, and 1.0mg metal/kg body weight. Lung O2 consumption was found to be significantly increased after the exposure to most doses of Ni-NP and Fe-NP, and the 0.05mg metal/kg body weight dose of Cr-NP, while no changes were observed for Cd-NP. Lucigenin chemiluminescence (as an indicator of NADPH oxidase (NOX) activity) was evaluated in lung homogenates. Only Ni-NP and Fe-NP have shown the ability to induce a significant increase in lucigenin chemiluminescence. In order to establish the possible occurrence of pulmonary oxidative stress, TBARS levels and the GSH/GSSG ratio were determined. The higher doses of Ni-NP and Fe-NP were able to induce an oxidative stress condition, as shown by changes in both TBARS levels and the GSH/GSSG ratio. Taken together, the present results show differential effects for all the metals tested. These findings emphasize the importance of transition metals present air PM in PM adverse health effects, and contribute to the understanding of the pathological mechanisms triggered by the exposure to environmental PM.