Ventricular remodeling of single-chambered myh6-/- adult zebrafish hearts occurs via a hyperplastic response and is accompanied by elastin deposition in the atrium.

Zebrafish (Danio rerio) is widely used as an animal model to understand the pathophysiology of cardiovascular diseases. Here, we present the adult cardiac phenotype of weak atrium, myh6-/-, which carry mutations in the zebrafish atrial myosin heavy chain. Homozygous mutants survive to adulthood and are fertile despite their initial weak atrial beat. In adult mutants, the atrium remains hypoplastic and shows elastin deposition while mutant ventricles exhibit increased size. In mammals, hypertrophy is the most common mechanism resulting in cardiomegaly. Using immunohistochemistry and confocal microscopy to measure cardiomyocyte cell size, density and proliferation, we show that the enlargement of the myh6-/- ventricle is predominantly due to hyperplasia. However, we identified similar transcriptional profiles to the mammalian hypertrophy response via RT-PCR of the hyperplastic ventricles. Furthermore, we show activation of the ER-stress pathway by western blot analysis. In conclusion, we can assume, based on our model, that molecular signaling pathways associated with hypertrophy in mammals, in combination with ER-stress activation, result in hyperplasia in zebrafish. In addition, to our knowledge, this is the first time to report elastin deposition in the atrium.

Evaluation of two novel antioxidants with differential effects on curcumin-induced apoptosis in C2 skeletal myoblasts; involvement of JNKs.

Excessive levels of reactive oxygen species (ROS) result in numerous pathologies including muscle disorders. In essence, skeletal muscle performance of daily activities can be severely affected by the redox imbalances occurring after muscular injuries, surgery, atrophy due to immobilization, dystrophy or eccentric muscle contraction. Therefore, research on the potential beneficial impact of antioxidants is of outmost importance. In this context, aiming at further exploring the mechanisms of action of our newly synthesized antioxidant compounds (AK1 and AK2) in a skeletal muscle experimental setting, we initially investigated their scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and subsequently assessed their effect on the viability of C2 skeletal myoblasts in the presence of two pro-oxidants: H2O2 and curcumin (MTT assay). Interestingly, while both compounds reversed the detrimental effect of H2O2, only AK2 was cytoprotective in curcumin-treated C2 cells. We next confirmed the immediate activation of extracellular signal-regulated kinases (ERKs) and the more delayed activation profile of c-Jun NH2-terminal kinases (JNKs) in C2 skeletal myoblasts exposed to curcumin, by Western blotting. In correlation with the aforementioned results, only AK2 blocked the curcumin-induced activation of JNKs pathway. Furthermore, JNKs were revealed to mediate curcumin-induced apoptosis in C2 cells and only AK2 to effectively suppress it (by detecting its effect on poly(ADP-ribose) polymerase fragmentation). Overall, we have shown that two similar in structure novel antioxidants confer differential effects on C2 skeletal myoblasts viability under oxidative stress conditions. This result may be attributed to these antioxidants respective diverse mode of interaction with the signaling effectors involved in the observed responses. Future studies should further evaluate the mechanism of action of these compounds in order to support their potential application in therapeutic protocols against ROS-related muscle disorders.

Curcumin induces the apoptotic intrinsic pathway via upregulation of reactive oxygen species and JNKs in H9c2 cardiac myoblasts.

Curcumin derived from the rhizome of turmeric (Curcuma longa L.), is a well known coloring culinary agent, that has therapeutic properties against diverse pathologies such as cancer, atherosclerosis and heart failure. Given the salutary potential of curcumin, deciphering its mode of action particularly in cardiac cells, is of outstanding value. Accumulating evidence implicates curcumin in the regulation of multiple signaling pathways leading to cell survival or apoptosis. Therefore, the present study aimed at elucidating the molecular mechanisms triggered by curcumin in H9c2 cells. Curcumin was found to activate p38-mitogen-activated protein kinase (p38-MAPK) as well as c-jun NH2 terminal kinases (JNKs), in a dose- and time-dependent manner. We also observed curcumin to impair cell survival by promoting apoptosis, evidenced by chromatin condensation, poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage, as well as Bax translocation and cytochrome c release into the cytosol. Curcumin-induced apoptosis was ascribed to JNKs, since hindering their activity abolished PARP fragmentation. Furthermore, we identified curcumin to exert a pro-oxidative activity, with 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining revealing up-regulation of reactive oxygen species (ROS) levels and anti-oxidants found to abrogate PARP cleavage. In conclusion, curcumin was found to stimulate the apoptotic cell death of H9c2 cells by upregulating ROS generation and triggering activation of JNKs. With reports underscoring the capacity of curcumin to perturb the cellular redox balance ensuring survival or enhancing apoptosis, determination of its mode of action appears to be critical. Future studies should assess the appropriate administration conditions of curcumin, so as to optimize its therapeutic potential against cardiovascular pathologies.

Phosphorylation of a p38-like MAPK is involved in sensing cellular redox state and drives atypical tubulin polymer assembly in angiosperms.

Reactive oxygen species (ROS) imbalance is a stressful condition for plant cells accompanied by dramatic changes in tubulin cytoskeleton. Here, evidence is provided that alterations in ROS levels directly interfere with the phosphorylation state of a p38-like MAPK in the angiosperms Triticum turgidum and Arabidopsis thaliana. Both oxidative stress generators and chemicals inducing ROS scavenging or decreasing ROS production resulted in the accumulation of a phospho-p46 protein similar to p38-MAPK. Importantly, the rhd2 A. thaliana mutants exhibited a remarkable increase in levels of phospho-p46. The presence of the p38-MAPK inhibitor SB203580 attenuated the response to ROS disturbance, prevented microtubule disappearance and resulted in a dramatic decrease in the number of atypical tubulin polymers. Moreover, in roots treated simultaneously with substances inducing ROS overproduction and others resulting in low ROS levels, phospho-p46 levels and the organization of tubulin cytoskeleton were similar to controls. Collectively, our experimental data suggest, for the first time in plants, that p46 functions as a putative sensor of redox state, the activation of which initiates downstream signalling events leading to microtubule disruption and subsequent assembly of atypical tubulin polymers. Thus, p46 seems to participate in perception of ROS homeostasis disturbance as well as in cellular responses to redox imbalance.

Calcium paradox induces apoptosis in the isolated perfused Rana ridibunda heart: involvement of p38-MAPK and calpain.

"Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

Severe Hyperosmotic Stress Issues an ER Stress-Mediated "Death Sentence" in H9c2 Cells, with p38-MAPK and Autophagy "Coming to the Rescue".

With several cardiovascular pathologies associated with osmotic perturbations, researchers are in pursuit of identifying the signaling sensors, mediators and effectors involved, aiming at formulating novel diagnostic and therapeutic strategies. In the present study, H9c2 cells were treated with 0.5 M sorbitol to elicit hyperosmotic stress. Immunoblotting as well as cell viability analyses revealed the simultaneous but independent triggering of multiple signaling pathways. In particular, our findings demonstrated the phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) and upregulation of the immunoglobulin heavy-chain-binding protein (BiP) expression, indicating the onset of the Integrated Stress Response (IRS) and endoplasmic reticulum stress (ERS), respectively. In addition, autophagy was also induced, evidenced by the enhancement of Beclin-1 protein expression and of AMP-dependent kinase (AMPK) and Raptor phosphorylation levels. The involvement of a Na+/H+ exchanger-1 (NHE-1) as well as NADPH oxidase (Nox) in 0.5 M sorbitol-induced eIF2α phosphorylation was also indicated. Of note, while inhibition of ERS partially alleviated the detrimental effect of 0.5 M sorbitol on H9c2 cellular viability, attenuation of p38-MAPK activity and late phase autophagy further mitigated it. Deciphering the mode of these pathways' potential interactions and of their complications may contribute to the quest for effective clinical interventions against associated cardiovascular diseases.

Differential roles of MAPKs and MSK1 signalling pathways in the regulation of c-Jun during phenylephrine-induced cardiac myocyte hypertrophy.

Gq-protein-coupled receptor (GqPCR) signalling is associated with the induction of cardiac myocyte hypertrophy, which is characterized by an increase in expression of immediate early genes via activation of pre-existing transcription factors. Here, we explore the role of MSK1 and MAPK signalling pathways in the regulation of the immediate early gene c-jun. The results provide further support for the role of MSK1 in cardiac myocyte hypertrophy and indicate that PE activates distinct signalling mechanisms which culminate with a complex activation of c-jun. ERK1/2 and JNKs are the principal kinases responsible for phosphorylation of c-Jun, whereas c-jun mRNA and protein up-regulation by PE is mediated by multiple signalling pathways that include MSK1, ERK1/2, p38-MAPK and JNKs. These signalling mechanisms seem to be critical to the phenotypic changes of cardiac myocytes in response to hypertrophic stimulation.

Oxidative stress and calpain inhibition induce alpha B-crystallin phosphorylation via p38-MAPK and calcium signalling pathways in H9c2 cells.

We investigated the response of alphaB-crystallin to oxidative stress and calpain inhibition in an attempt to elucidate the signalling pathways mediating its phosphorylation. Given the high expression levels of alphaB-crystallin in cardiac muscle one can evaluate the significance of its participation in preservation of homeostasis under adverse conditions. H9c2 cardiac myoblasts were used as our experimental model since their response reflects the signal transduction pathways activated by stress conditions in the myocardium. Thus, in H9c2 cells treated with H2O2 the mechanism regulating alphaB-crystallin phosphorylation was found to involve p38-MAPK/MSK1 as well as intracellular free calcium levels. Our immunocytochemical experiments demonstrated phosphorylated alphaB-crystallin to be co-localized with tubulin, potentially preserving cytoskeletal architecture under these interventions. In H9c2 cells treated with calpain inhibitors (ALLN, ALLM) alphaB-crystallin exhibited a p38-MAPK- and [Ca 2+](i)-dependent phosphorylation pattern since the latter was ablated in the presence of the selective p38-MAPK inhibitor SB203580 and calcium chelator BAPTA-AM. Calpain activity repression ultimately led to apoptosis confirmed by PARP fragmentation and chromatin condensation. However, the apoptotic pathway activated by ALLM and ALLN differed, underlying the diverse transduction mechanisms stimulated. In addition to this, an anti-apoptotic role for phospho-alphaB-crystallin was verified by confirmation of its interaction with pro-caspase 3, hindering its cleavage and subsequent activation. Collectively, our findings underline alphaB-crystallin crucial role as a participant of cardiac cells early response to stressful stimuli compromising their survival.

p38-MAPK is involved in restoration of the lost protection of preconditioning by nicorandil in vivo.

Nicorandil, a selective mitochondrial K(ATP) channel opener, reinstates the waned protection after multiple cycles of preconditioning. In this study, we determined the signal transduction activated in heart after 3 or 8 cycles of preconditioning and prolonged ischemia in rabbits treated with placebo or nicorandil. In a first series (eight groups) we evaluated the (%) infarct to risk ratio after 30 min ischemia/3 h reperfusion and in a second series (six groups), we assessed the intracellular levels of cyclic GMP (c-GMP), protein kinase C (PKC) activity and p38-mitogen activated protein kinase (p38-MAPK) phosphorylation from heart samples taken during the long ischemia. Cardioprotection by 3 cycles of preconditioning (11.7+/-3.8% vs 45.9+/-5.2% in the control, P<0.001) was lost after 8 cycles (43.9+/-5.1%, P=NS vs control). Nicorandil restored it to the levels of classic preconditioning (13.7+/-2.4% vs 40.8+/-3.5% in respective controls, P<0.001). This was reversed by the p38-MAPK inhibitor SB203580 (48.8+/-5.1%) which had no protective effect in the control group (44.6+/-5.8%). In the placebo-treated rabbits, intracellular c-GMP and PKC were increased only in the group subjected to 3 cycles of preconditioning. Despite that nicorandil equalizes the intracellular levels of c-GMP, PKC and activated p38-MAPK at the long ischemia, specific alterations of p38-MAPK phosphorylation differentiate the protected groups. Our data delineate the signal transduction mechanism mediating the beneficial effect of nicorandil and imply that the recapture of the lost protection is due to a dynamic process of the intracellular mediators accompanied by an increase in p38-MAPK phosphorylation and not to an instantaneous event.

Induction of protective cellular immune responses against experimental visceral leishmaniasis mediated by dendritic cells pulsed with the N-terminal domain of Leishmania infantum elongation factor-2 and CpG oligodeoxynucleotides.

Leishmania elongation factor 2 (EF-2) has been previously identified as a TH1-stimulatory protein. In this study, we assayed the protective potential of the N-terminal domain of EF-2 (N-LiEF-2, 1-357 aa) that has been predicted to contain several overlapping MHC class I and II-restricted epitopes injected in the form of dendritic cell (DC)-based vaccine. Ex vivo pulsing of DCs with the recombinant N-LiEF-2 domain along with CpG oligodeoxynucleotides (ODNs) resulted in their functional differentiation. BALB/c vaccinated with CpG-triggered DCs pulsed with N-LiEF-2 were found to be the most immune-reactive in terms of induction of DTH responses, increased T cell proliferation and IL-2 production. Moreover, vaccination induced antigen-specific TH1 type immune response as evidenced by increased IFN-γ and TNFα levels followed by a significant increase of nitrite (NO) and reactive oxygen species (ROS) in splenocyte cultures. Vaccinated mice showed a pronounced decrease in parasite load in spleen and liver when challenged with L. infantum, increased expression of Stat1 and Tbx21 mRNA transcripts versus reduced expression of Foxp3 transcripts and were able to produce significantly elevated levels of IL-2, IFN-γ and TNFα but not IL-10 compared to non-vaccinated mice. Both antigen and parasite-specific CD4+ T and CD8+ T cells contributed to the IFN-γ production indicating that both subtypes contribute to the resistance to infection and correlated with robust nitrite generation, critical in controlling Leishmania infection. Together, these findings demonstrated the immunogenic as well as protective potential of the N-terminal domain of Leishmania EF-2 when given with CpG-triggered DCs representing a basis for the development of rationalized vaccine against leishmaniasis.

ERK1/2 and p38-MAPK signalling pathways, through MSK1, are involved in NF-kappaB transactivation during oxidative stress in skeletal myoblasts.

Skeletal muscle is highly adapted to respond to oxidative imbalances, since it is continuously subjected to an increased production of reactive oxygen species (ROS) during exercise. Oxidative stress, however, has been associated with skeletal muscle atrophy and damage in many diseases. In this study, we examined whether MAPK and NF-kappaB pathways participate in the response of skeletal myoblasts to oxidative stress, and whether there is a cross talk between these pathways. H(2)O(2) induced a strong activation of ERKs, JNKs and p38-MAPK in a time- and dose-dependent profile. ERK and JNK activation by H(2)O(2), but not that of p38-MAPK, was mediated by Src kinase and, at least in part, by EGFR. H(2)O(2) also stimulated a mild translocation of NF-kappaB to the nucleus, as well as a moderate phosphorylation of its endogenous cytoplasmic inhibitor IkappaB (at Ser32/36), without any significant decrease in IkappaB total levels. Moreover, oxidative stress induced a strong phosphorylation of NF-kappaB p65 subunit at Ser536 and Ser276. Inhibition of MAPK pathways by selective inhibitors did not appear to affect H(2)O(2)-induced nuclear translocation of NF-kappaB or the phosphorylation of IkappaB. In contrast, phosphorylation of p65 at Ser276 was found to be mediated by MSK1, a substrate of both ERKs and p38-MAPK. In conclusion, it seems that, during oxidative stress, NF-kappaB translocation to the nucleus is most likely not related with the MAPK activation, while p65 phosphorylations are in part mediated by MAPKs pathways, probably modifying signal specificity.

Involvement of JNKs and p38-MAPK/MSK1 pathways in H2O2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells.

One of the most important challenges that cardiomyocytes experience is an increase in the levels of reactive oxygen species (ROS), i.e., during ischemia, reperfusion as well as in the failing myocardium. HOX-1 has been found to protect cells and tissues against oxidative damage; therefore, we decided to study the signalling cascades involved in its transcriptional regulation. HOX-1 mRNA levels were found to be maximally induced after 6h of treatment with 200 microM H2O2 and remained elevated for at least 24h. Inhibition of JNKs, p38-MAPK and MSK1 pathways, by pharmacological inhibitors, reduced HOX-1 mRNA levels in H2O2-treated H9c2 cells. In parallel, we observed that all three subfamilies of the mitogen-activated protein kinases (MAPKs) attained their maximal phosphorylation levels at 5-15 min of H2O2 treatment, with mitogen- and stress-activated-protein kinase 1 (MSK1) also being maximally phosphorylated at 15 min. H2O2-induced MSK1 phosphorylation was completely abrogated in the presence of the selective p38-MAPK inhibitor SB203580. In an effort to define possible substrates of MSK1, we found that ATF2 as well as cJun phosphorylation were equally induced after 30 min and 60 min, respectively, a response inhibited by SP600125 (JNKs inhibitor) and H89 (MSK1 inhibitor), indicating the involvement of these kinases in the observed response. This finding was further substantiated with the detection of a potential signalling complex composed of either p-MSK1 and p-cJun or p-MSK1 and p-ATF2 (co-immunoprecipitation). ATF2 and cJun are known AP1 components. Given the presence of an AP-1 site in HOX-1 promoter region, the activity of AP1 transcription factor was examined. Electrophoretic mobility shift assays performed showed a maximal upregulation of AP1 binding activity after 60 min of H2O2 treatment, which was significantly inhibited by SP600125 and H89. Our results show for the first time the potential role of JNKs, p38-MAPK and MSK1 in the mechanism of transducing the oxidative stress-signal to HOX-1, possibly promoting cell survival and preserving homeostasis.

Vaccination with poly(D,L-lactide-co-glycolide) nanoparticles loaded with soluble Leishmania antigens and modified with a TNFα-mimicking peptide or monophosphoryl lipid A confers protection against experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) persists as a major public health problem, and since the existing chemotherapy is far from satisfactory, development of an effective vaccine emerges as the most appropriate strategy for confronting VL. The development of an effective vaccine relies on the selection of the appropriate antigen and also the right adjuvant and/or delivery vehicle. In the present study, the protective efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which were surface-modified with a TNFα-mimicking eight-amino-acid peptide (p8) and further functionalized by encapsulating soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand, was evaluated against challenge with L. infantum parasites in BALB/c mice. Vaccination with these multifunctionalized PLGA nanoformulations conferred significant protection against parasite infection in vaccinated mice. In particular, vaccination with PLGA-sLiAg-MPLA or p8-PLGA-sLiAg NPs resulted in almost complete elimination of the parasite in the spleen for up to 4 months post-challenge. Parasite burden reduction was accompanied by antigen-specific humoral and cellular immune responses. Specifically, injection with PLGA-sLiAg-MPLA raised exclusively anti-sLiAg IgG1 antibodies post-vaccination, while in p8-PLGA-sLiAg-vaccinated mice, no antibody production was detected. However, 4 months post-challenge, in mice vaccinated with all the multifunctionalized NPs, antibody class switching towards IgG2a subtype was observed. The study of cellular immune responses revealed the increased proliferation capacity of spleen cells against sLiAg, consisting of IFNγ-producing CD4+ and CD8+ T cells. Importantly, the activation of CD8+ T cells was exclusively attributed to vaccination with PLGA NPs surface-modified with the p8 peptide. Moreover, characterization of cytokine production in vaccinated-infected mice revealed that protection was accompanied by significant increase of IFNγ and lower levels of IL-4 and IL-10 in protected mice when compared to control infected group. Conclusively, the above nanoformulations hold promise for future vaccination strategies against VL.

Acute administration of vitamin E triggers preconditioning via K(ATP) channels and cyclic-GMP without inhibiting lipid peroxidation.

Vitamin E (VitE) is considered an antioxidant agent. One or more brief periods of ischemia (isc), followed by short reperfusion (rep), increase the tolerance of the heart to a subsequent prolonged ischemia, a phenomenon known as ischemic preconditioning (PC). Mitochondrial KATP channels (mitoKATP), cyclic-GMP (cGMP), and free radicals are involved in the mechanism of PC, whereas some antioxidants abolish this benefit. The purpose of this study was to evaluate the effect of VitE on infarct size, PC, and the oxidative status in vivo. Male rabbits were divided into seven groups and were subjected to myocardial ischemia (isc) and reperfusion (rep) with the following interventions: (1) control (no intervention); (2) E150 (iv VitE at a dose of 150 mg/kg for 75 min, starting 40 min before index isc and lasting through 5 min of rep); (3) E300 (iv VitE 300 mg/kg as previously described); (4) PC (two cycles of 5 min isc and 10 min rep), (5) combined E150-PC; and (6) combined E300-PC. In the last two groups VitE was given 40 min before index ischemia. Blood samples were taken for malondialdehyde (MDA) and conjugated dienes (CDs) measurement. In a second series of experiments heart tissue samples were taken at the time of long ischemia for MDA and CD determination and for cGMP assay. In order to test whether combined treatment with VitE (as the E150 group) and the mitoKATP blocker 5-hydroxydecanoic acid (5-HD) changes the infarct size, an additional group was assessed in the first series of experiments. Tissue VitE concentration was evaluated in myocardium. VitE at both doses reduced the infarct size (19.7 +/- 2.8% for E150 and 18.8 +/- 4.9% for E300 vs 47.4 +/- 2.6% in control, P < 0.05) without attenuating the effect of PC (10.2 +/- 3.1% for E150-PC, 12.4 +/- 2.2% for E300-PC, vs 13.5 +/- 3.3% for PC). Combined VitE and 5-HD treatment abrogates this benefit (37.4 +/- 6.5%, P < 0.05 vs E150 and NS vs control). VitE increases intracellular cGMP and CDs levels (P < 0.05 vs control) to the same extent as PC (P < 0.05 vs control), with no effect on MDA (P = NS between all the groups). Peripheral markers of oxidative stress are increased during reperfusion in all groups (P < 0.05 vs baseline). Overall, VitE limits infarct size via mitoKATP and cGMP, while preserving the benefit of ischemic PC.

PLGA nanoparticles modified with a TNFα mimicking peptide, soluble Leishmania antigens and MPLA induce T cell priming in vitro via dendritic cell functional differentiation.

Poly(lactide-co-glycolide) nanoparticles (PLGA NPs) represent a new approach for vaccine delivery due to their ability to be taken up by phagocytes and to activate immune responses. In the present study PLGA NPs were surface-modified with a TNFα mimicking peptide, and encapsulated soluble Leishmania antigens (sLiAg) and MPLA adjuvant. The synthesized PLGA NPs exhibited low cytotoxicity levels, while surface-modified NPs were more efficiently taken up by dendritic cells (DCs). The prepared nanoformulations induced maturation and functional differentiation of DCs by elevating co-stimulatory molecule levels and stimulating IL-12 and IL-10 production. Sensitized DCs promoted T cell priming, characterized by the development of mixed T cell subsets differentiation expressing Th lineage-specific transcriptional factors and cytokine genes. Moreover, PLGA NPs were biocompatible, while they were located in lymphoid organs and taken up by phagocytic cells. Our results suggest that surface-modified PLGA NPs encapsulating sLiAg and MPLA could be considered as an effective vaccine candidate against leishmaniasis.

Curcumin acts as a pro-oxidant inducing apoptosis via JNKs in the isolated perfused Rana ridibunda heart.

Amphibians are known to better tolerate and endure adverse environmental conditions such as redox imbalances conferred by reactive oxygen species (ROS), compared to mammals. Interestingly, the exact adaptation strategies and signaling mechanisms mediating these effects have not been fully elucidated. Therefore, in the present study, we probed into the molecular response of the isolated perfused Rana ridibunda heart to curcumin, in the context of mitogen-activated protein kinases (MAPKs) phosphorylation patterns and apoptotic markers occurrence. In particular, this polyphenol was found to exert a pro-oxidant effect in our model and to significantly upregulate p38-MAPK and JNKs phosphorylation (thus activation). The early apoptosis observed, substantiated by poly(ADP-ribose) polymerase (PARP) cleavage, was established to be JNKs- and ROS-mediated, while no involvement of p38-MAPK was detected. Subsequently, the pro-oxidative activity of curcumin was confirmed to mimic H(2) O(2). Furthermore, NADPH oxidase as well as Na(+) /K(+) -ATPase were found to mediate JNKs phosphorylation as well as PARP proteolytic cleavage. Curcumin exerts pleiotropic actions, both beneficial and detrimental and is currently the subject of intense scientific research. Being a low-molecular-weight antioxidant, it is intriguing to investigate curcumin's role in redox homeostasis in the amphibian heart, under conditions that apparently favor its pro-oxidative properties. Comparative studies of its multifaceted role in different species may contribute to the clarification of the signaling mechanisms it triggers and the terminal physiological response it confers. Collectively, this is to our knowledge, the first time that the signal transduction pathways stimulated by curcumin have been assessed in a non-mammalian species.

Ovariectomy reinstates the infarct size-limiting effect of postconditioning in female rabbits.

Gender seems to interfere with the cardioprotective effect of ischemic preconditioning (PreC) and postconditioning (PostC); PreC-conferred protection is weaker or lost in female animals after ovariectomy (Ov), while the role of PostC is still in dispute. We sought to investigate the effect of PostC in female rabbits, its interaction with Ov, and the potential implicated intracellular pathways. Intact or Ov adult female rabbits (n = 46) were subjected to 30 min ischemia and reperfusion with PostC (PostC or OvPostC), which consisted of six cycles of 30-s ischemia/30-s reperfusion at the end of ischemia, or without PostC (Fem or OvFem). Infarct size (I) and area at risk (R) were determined by TTC staining and fluorescent particles, respectively, after 3-h reperfusion in 30 out of 46 animals. Plasma levels of estradiol and nitrite/nitrate (NO x ) were evaluated. ERKs, p38-MAPK, and Akt assessment was performed in excised hearts 1-min after starting the final reperfusion period in the remaining 16 animals. Infarct size was significantly reduced only in OvPostC group (I/R ratio, 25.3 ± 2.7, vs 48.1 ± 2.0, 43.6 ± 4.2 and 55.1 ± 5.6 % in Fem, OvFem, and PostC groups, p < 0.05). In ovariectomized rabbits, plasma estradiol and NO x levels were lower than in the normal ones. Akt phosphorylation in ischemic regions was significantly higher in OvPostC group, whereas ERK1/2 and p38-MAPK activation was observed in all ovariectomized animals irrespective of PostC. PostC is not effective in female rabbits, but the protection is reinstated after Ov potentially via the RISK pathway.

Insulin-induced oxidative stress up-regulates heme oxygenase-1 via diverse signaling cascades in the C2 skeletal myoblast cell line.

Impaired insulin sensitivity (insulin resistance) is a common denominator in many metabolic disorders, exerting pleiotropic effects on skeletal muscle, liver, and adipose tissue function. Heme oxygenase-1 (HOX-1), the rate-limiting enzyme in heme catabolism, has recently been shown to confer an antidiabetic effect while regulating cellular redox-buffering capacity. Therefore, in the present study, we probed into the mechanisms underlying the effect of insulin on HOX-1 in C2 skeletal myoblasts. Hence, insulin was found to suppress C2 myoblasts viability via stimulation of oxidative stress, with HOX-1 counteracting this action. Insulin induced HOX-1 expression in a time- and dose-dependent manner, an effect attenuated by selective inhibitors of ERK1/2 (PD98059), Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and c-Jun terminal kinases 1 and 2 (SP600125) pathways. Furthermore, nuclear factor-κB role in insulin-induced HOX-1 up-regulation was verified, with ERK1/2, Src, and c-Jun terminal kinases 1 and 2 mediating p65-nuclear factor-κB subunit phosphorylation. Overall, our novel findings highlight for the first time the transduction mechanisms mediating HOX-1 induction in insulin-treated C2 myoblasts. This effect was established to be cell type specific because insulin failed to promote HOX-1 expression in HepG2 hepatoma cells. Deciphering the signaling networks involved in insulin-stimulated HOX-1 up-regulation is of prominent significance because it may potentially contribute to elucidation of the mechanisms involved in associated metabolic pathologies.

HOX-1 and COX-2: Two differentially regulated key mediators of skeletal myoblast tolerance under oxidative stress.

The exact physiological role of oxidative stress as a primary cause for skeletal muscle pathological conditions involving muscle degeneration remains elusive. Therefore, the present study was performed so as to decipher the signalling pathways orchestrating the potential cytoprotective role of heme oxygenase 1 (HOX-1) as well as cyclooxygenase 2 (COX-2) in skeletal myoblasts exposed to H(2)O(2). Cell treatment with H(2)O(2) (0.5 mM) resulted in a time- and dose-dependent response of HOX-1 and COX-2 mRNA and protein levels, with ERK1/2, p38-MAPK and MSK1 found to mediate these effects. Furthermore, Src and JNKs blockade attenuated COX-2 response. Collectively, these novel findings highlight for the first time HOX-1 and COX-2 fundamental contribution to skeletal myoblast tolerance under oxidative stress, since their inhibition significantly attenuated viability of skeletal myoblasts. The data also delineate the various effectors regulating HOX-1 and COX-2 expression, probably alleviating muscle degeneration in related disorders.

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells.

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H2O2, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H2O2 stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 microM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.