Actin-Cytoskeleton Drives Caveolae Signaling to Mitochondria during Postconditioning.

Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.

Implications of Oxidative and Nitrosative Post-Translational Modifications in Therapeutic Strategies against Reperfusion Damage.

Post-translational modifications based on redox reactions "switch on-off" the biological activity of different downstream targets, modifying a myriad of processes and providing an efficient mechanism for signaling regulation in physiological and pathological conditions. Such modifications depend on the generation of redox components, such as reactive oxygen species and nitric oxide. Therefore, as the oxidative or nitrosative milieu prevailing in the reperfused heart is determinant for protective signaling, in this review we defined the impact of redox-based post-translational modifications resulting from either oxidative/nitrosative signaling or oxidative/nitrosative stress that occurs during reperfusion damage. The role that cardioprotective conditioning strategies have had to establish that such changes occur at different subcellular levels, particularly in mitochondria, is also presented. Another section is devoted to the possible mechanism of signal delivering of modified proteins. Finally, we discuss the possible efficacy of redox-based therapeutic strategies against reperfusion damage.

Hepatocyte growth factor enhances the clearance of a multidrug-resistant Mycobacterium tuberculosis strain by high doses of conventional chemotherapy, preserving liver function.

Tuberculosis (TB) is one of the deadliest infectious diseases in humankind history. Although, drug sensible TB is slowly decreasing, at present the rise of TB cases produced by multidrug-resistant (MDR) and extensively drug-resistant strains is a big challenge. Thus, looking for new therapeutic options against these MDR strains is mandatory. In the present work, we studied, in BALB/c mice infected with MDR strain, the therapeutic effect of supra-pharmacological doses of the conventional primary antibiotics rifampicin and isoniazid (administrated by gavage or intratracheal routes), in combination with recombinant human hepatocyte growth factor (HGF). This high dose of antibiotics administered for 3 months, overcome the resistant threshold of the MDR strain producing a significant reduction of pulmonary bacillary loads but induced liver damage, which was totally prevented by the administration of HGF. To address the long-term efficiency of this combined treatment, groups of animals after 1 month of treatment termination were immunosuppressed by glucocorticoid administration and, after 1 month, mice were euthanized, and the bacillary load was determined in lungs. In comparison with animals treated only with a high dose of antibiotics, animals that received the combined treatment showed significantly lower bacterial burdens. Thus, treatment of MDR-TB with very high doses of primary antibiotics particularly administrated by aerial route can produce a very good therapeutic effect, and its hepatic toxicity can be prevented by the administration of HGF, becoming in a new treatment modality for MDR-TB.

Cadmium exposure exacerbates hyperlipidemia in cholesterol-overloaded hepatocytes via autophagy dysregulation.

Metabolic factors are the major risk of non-alcoholic fatty liver disease, although other factors may contribute steatosis. Cadmium exposure produces histopathological and molecular changes in liver, which are consistent with steatosis. In the present study, we describe the effect of low cadmium acute treatment on hepatocytes obtained from mice fed with a high cholesterol diet. Our data suggest that hepatocytes with cholesterol overload promote an adaptive response against cadmium-induced acute toxicity by up-regulating anti-apoptotic proteins, managing ROS overproduction, increasing GSH synthesis and MT-II content to avoid protein oxidation. Cadmium treatment increases lipid content in cholesterol-fed mice hepatocytes because of an impaired autophagy process. Our data suggest an essential function of macroautophagy in the regulation of lipid storage induced by Cd on hepatocytes, that implies that alterations in this pathway may be a mechanism that aggravates hepatic steatosis.

Cholesterol overload in the liver aggravates oxidative stress-mediated DNA damage and accelerates hepatocarcinogenesis.

Primary liver cancers represent the second leading cause of cancer-related deaths worldwide. Diverse etiological factors include chronic viral hepatitis, aflatoxin and alcohol exposure as well as aberrant liver lipid overload. Cholesterol has been identified as a key inducer of metabolic impairment, oxidative stress and promoter of cellular dysfunction. The aim of this work was to address the oxidative stress-mediated DNA damage induced by cholesterol overload, and its role in the development of hepatocellular carcinoma. C57BL/6 male mice were fed with a high cholesterol diet, followed by a single dose of N-diethylnitrosamine (DEN, 10 µg/g, ip). Reactive oxygen species generation, DNA oxidation, antioxidant and DNA repair proteins were analyzed at different time points. Diet-induced cholesterol overload caused enhanced oxidative DNA damage in the liver and was associated with a decrease in key DNA repair genes as early as 7 days. Interestingly, we found a cell survival response, induced by cholesterol, judged by a decrement in Bax to Bcl2 ratio. Importantly, N-acetyl-cysteine supplementation significantly prevented DNA oxidation damage. Furthermore, at 8 months after DEN administration, tumor growth was significantly enhanced in mice under cholesterol diet in comparison to control animals. Together, these results suggest that cholesterol overload exerts an oxidative stress-mediated effects and promotes the development of liver cancer.

Hepatocyte growth factor protects against isoniazid/rifampicin-induced oxidative liver damage.

The worldwide increment of multidrug- and extensively drug-resistant tuberculosis has emphasized the importance of looking for new options in therapeutics. Long-time usage or higher doses of isoniazid and rifampicin have been considered for the treatment of multidrug-resistant tuberculosis; however, the risk of liver failure is proportionally increased. Hepatocyte growth factor (HGF) is a multitask growth factor that stimulates both antiapoptotic and antioxidant responses that counteract the toxic effects of drug metabolism in the liver. The present work was focused to address the antioxidant and antiapoptotic effects of HGF on isoniazid- and rifampicin-induced hepatotoxicity. BALB/c mice were subjected to rifampicin (150mg/kg, intragavage [ig]) plus isoniazid (75mg/kg, ig) for 7 days. Increments in alanine aminotransferase activity, steatosis, apoptosis, and oxidative stress markers were found in animals. Recombinant HGF (iv) prevented all the harmful effects by increasing the activation of Erk1/2 and PKCδ signaling pathways and glutathione (GSH) synthesis. Furthermore, inhibition of endogenous HGF with anti-HGF antibody (iv) enhanced the isoniazid- and rifampicin-induced oxidative stress damage and decreased the GSH content, aggravating liver damage. In conclusion, HGF demonstrated to be a good protective factor against antituberculosis drug-induced hepatotoxicity and could be considered a good adjuvant factor for the use of high doses of or the reintroduction of these antituberculosis drugs.

Biphasic regulation of the NADPH oxidase by HGF/c-Met signaling pathway in primary mouse hepatocytes.

Redox signaling is emerging as an essential mechanism in the regulation of biological activities of the cell. The HGF/c-Met signaling pathway has been implicated as a key regulator of the cellular redox homeostasis and oxidative stress. We previously demonstrated that genetic deletion of c-Met in hepatocytes disrupts redox homeostasis by a mechanism involving NADPH oxidase. Here, we were focused to address the mechanism of NADPH oxidase regulation by HGF/c-Met signaling in primary mouse hepatocytes and its relevance. HGF induced a biphasic mechanism of NADPH oxidase regulation. The first phase employed the rapid increase in production of ROS as signaling effectors to activate the Nrf2-mediated protective response resulting in up-regulation of the antioxidant proteins, such as NAD(P)H quinone oxidoreductase and γ-glutamylcysteine synthetase. The second phase operated under a prolonged HGF exposure, caused a suppression of the NADPH oxidase components, including NOX2, NOX4, p22 and p67, and was able to abrogate the TGFß-induced ROS production and improve cell viability. In conclusion, HGF/c-Met induces a Nrf2-mediated protective response by a double mechanism driven by NADPH oxidase.