Biomedical subjects

Anastasis Stephanou

Publications and source records attributed to Anastasis Stephanou.

29 records · Page 2Linked to original sources

PubMed2003

STAT-1 interacts with p53 to enhance DNA damage-induced apoptosis.

The STAT-1 transcription factor has been implicated as a tumor suppressor by virtue of its ability to inhibit cell growth and promoting apoptosis. However, the mechanisms by which STAT-1 mediates these effects remain unclear. Using human and mouse STAT-1-deficient cells, we show here that STAT-1 is required for optimal DNA damage-induced apoptosis. The basal level of the p53 inhibitor Mdm2 is increased in STAT-1(-/-) cells, suggesting that STAT-1 is a negative regulator of Mdm2 expression. Correspondingly, both basal p53 levels, and those induced by DNA damage were lower in STAT-1(-/-) cells. In agreement with this lower p53 response to DNA damage in cells lacking STAT-1, the induction of p53 responsive genes, such as Bax, Noxa, and Fas, was reduced in STAT-1-deficient cells. Conversely, STAT-1 overexpression enhances transcription of these genes, an effect that is abolished if the p53 response element in their promoters is mutated. Moreover, STAT-1 interacts directly with p53, an association, which is enhanced following DNA damage. Therefore, in addition to negatively regulating Mdm2, STAT-1 also acts as a coactivator for p53. Hence STAT-1 is another member of a growing family of protein partners able to modulate the p53-activated apoptotic pathway.

Animals↗

PubMed2003

FLIP protects cardiomyocytes from apoptosis induced by simulated ischemia/reoxygenation, as demonstrated by short hairpin-induced (shRNA) silencing of FLIP mRNA.

Cardiomyocytes exposed to ischemia followed by reperfusion undergo apoptosis, some of which is induced via the mitochondrial pathway, and some of which is induced via the death-receptor ligand pathway. FLICE-inhibitory protein (FLIP) is a cellular protein that, in overexpression experiments in other cell types, has been shown to be capable of either inducing or protecting from death-receptor-mediated apoptosis, depending on the extent of overexpression. To examine the role of endogenous FLIP in neonatal cardiomyocytes we have used short hairpin-induced (shRNA) silencing generated from a transfected plasmid. Silencing of FLIP increases the background level of apoptosis detected by TUNEL assay in primary cardiomyocytes and sensitizes the cells to apoptosis induced by simulated ischemia/reoxygenation (IR). This result indicates that previous observations of decreased FLIP levels and increased apoptosis in cardiac infarcts is more than just correlative, but reflects an innate protective role of FLIP in cardiomyocytes. We also found that mitochondrial activity, as determined by MitoTracker red staining, is reduced in the absence of FLIP, and further decreases after exposure to simulated IR. Conversely, overexpression of FLIP somewhat prevents the decrease in mitochondrial activity after simulated IR. Thus, FLIP may confer protection from simulated ischemia/reperfusion through more than one pathway.

Animals↗

PubMed2003

STAT-1: a novel regulator of apoptosis.

Extracellular signalling molecules binding to their specific receptors are able to modulate gene expression, leading to changes in development, cell growth and homeostasis. The signal transducers and activators of transcription (STAT) protein family members are among the best studied of the latent cytoplasmic signal-dependent transcription factors. The STAT factors are activated via phosphorylation on the C-terminal domain following cytokine signalling or by various stress-induced stimuli. Recently, STAT-1 has been implicated in modulating pro- and anti-apoptotic genes following several stress-induced responses. These effects are dependent on STAT-1 phosphorylation on serine-727 and require the C-terminal transactivation domain of STAT-1 to enhance its pro-apoptotic effect or inhibit its anti-apoptotic effects. The STAT-1 C-terminal domain has been demonstrated to be important for protein-protein interaction with other transcriptional activators. The reports that STAT-1-deficient mice develop spontaneous and chemically induced tumours more rapidly compared to wild-type mice and that STAT-1-deficient cells are more resistant to agents that induce apoptosis strongly support the argument that STAT-1 acts as a tumour suppressor.

Apoptosis↗

PubMed2002

The carboxyl-terminal activation domain of the STAT-1 transcription factor enhances ischemia/reperfusion-induced apoptosis in cardiac myocytes.

We have demonstrated previously that the STAT-1 transcription factor plays a key role in ischemia/reperfusion (I/R)-induced apoptosis in cardiac myocytes. In the present study we assessed which region of the STAT-1 molecule mediates apoptosis in cardiac myocytes. A STAT-1 construct (amino acid 350-750) lacking the N-terminus could enhance I/R-induced apoptosis in cardiac myocytes. However, a STAT-1 construct, which lacks 60 amino acids at the C-terminus (amino acid 691-750), was ineffective in promoting I/R-induced apoptosis in cardiac myocytes. Furthermore, overexpression of a C-terminal STAT-1 construct (amino acid 691-750) containing the transcriptional activation domain, but not the DNA binding domain, strongly enhanced I/R-induced apoptotic cell death. Cardiac myocytes isolated from mice expressing a truncated C-terminal STAT-1 were more sensitive to I/R-induced cell death. Finally, isolated hearts from these animals exposed to I/R injury had larger infarct size and greater number of TUNEL-positive myocytes than control hearts. These studies demonstrate that the C-terminal transactivation domain of STAT-1 is necessary and sufficient for I/R injury-induced apoptosis in cardiac myocytes.

Animals↗

PubMed2002

Urocortin promotes hemodynamic and bioenergetic recovery and improves cell survival in the isolated rat heart exposed to ischemia/reperfusion.

OBJECTIVES: This study evaluates the hemodynamic, bioenergetic and cytoprotective effects of urocortin (Ucn) in the isolated rat heart exposed to ischemia (I)/reperfusion (R). BACKGROUND: We have previously demonstrated that administration of exogenous Ucn reduces infarct size in ischemic-reperfused rat hearts. METHODS: Urocortin 10(-8)M was added to the perfusate before I, before I and during R, and during R alone in the isolated pulsed rat heart exposed to 35 min I followed by 60 min R. RESULTS: Partial to complete recovery of diastolic pressure and developed pressure was seen irrespective of when Ucn was perfused. In particular, beneficial effects are observed when Ucn is only given during R. Urocortin given only before I, and before I and over R, although not during R alone, also produces significant recovery of high-energy phosphate pools. In each group, improvement in ventricular function is associated with reduction both in myocardial damage, assessed by creatine phosphokinase release, and in endothelial cell and cardiomyocyte apoptosis, assessed by caspase 3 activity and fluorescent-based terminal deoxynucleotidyl transferase mediated nick end labelling enhanced with counterstains. These improvements in ventricular performance, bioenergetics and cell survival are not secondary to any inotropic effects of Ucn. CONCLUSIONS: This is the first report to show enhanced cardiac function induced by Ucn during I/R. Because the cytoprotective and functional benefits are still produced when Ucn is given only at R, these data suggest that Ucn may be useful clinically in the management of myocardial infarction.

Adenosine Triphosphate↗

PubMed2002

Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes during ischemia/reperfusion injury.

Apoptosis contributes, with necrosis, to the cardiac cell loss after ischemia/reperfusion injury. The apoptotic cascade is initiated either by mitochondrial damage and activation of caspase-9 or by death receptor ligation and activation of caspase-8. In the present study, performed in the isolated rat heart exposed either to ischemia alone or ischemia followed by reperfusion, cleavage of caspase-9 was observed primarily in endothelial cells. Conversely, caspase-8 cleavage was only found in cardiomyocytes, where it progressively increased throughout reperfusion. Addition of a specific caspase-9 inhibitor to the perfusate before ischemia prevented endothelial apoptosis, whereas preischemic infusion of a specific caspase-8 inhibitor affected only myocyte apoptosis. Additionally, caspase-8-mediated BID processing was observed only during reperfusion. Production of tBID then sustains mitochondrial injury and perpetuates caspase-9 activation.

Animals↗

PubMed2002

Cardiotrophin-1 and urocortin cause protection by the same pathway and hypertrophy via distinct pathways in cardiac myocytes.

Cardiotrophin-1 (CT-1) is an Interleukin-6 family cytokine with known hypertrophic and protective effects in cardiac cells. CT-1 and the corticotrophin releasing hormone-like hormone urocortin protect cardiac myocytes by the same p42/44 mitogen activated protein kinase (p42/44 MAPK) dependent pathway. We investigated whether urocortin is also hypertrophic in cardiac myocytes and whether it shares a common pathway with CT-1 for this effect. Moreover, we also investigated, for the first time whether CT-1 and urocortin can induce hypertrophy in cultured adult as opposed to neonatal cardiac cells. Urocortin and CT-1 caused hypertrophy (as measured by an increase in cell area and enhanced protein: DNA ratio) in both adult and neonatal rat cultured cardiac myocytes. The hypertrophic effect of CT-1 was dependent on the signal transducer and activator of transcription 3 (STAT3) pathway but the hypertrophic effect of urocortin was independent of this pathway. In contrast, inhibition of the protective p42/p44 MAPK pathway has no effect on the hypertrophic effect of CT-1 or urocortin. Additionally, inhibition of the STAT3 pathway has no effect on the protective effect of CT-1 or urocortin. These results identify urocortin as a novel hypertrophic and protective agent whose hypertrophic effect is mediated by a distinct pathway to that activated by CT-1, although the two factors mediate protection via the same pathway.

Animals↗

PubMed2002

Antiapoptotic activity of the free caspase recruitment domain of procaspase-9: a novel endogenous rescue pathway in cell death.

Mitochondrial injury initiates proteolytic processing of procaspase-9 into the large and small subunits, leading to apoptotic cell death. Here we show that the free caspase recruitment domain (CARD) released by procaspase-9 processing activates nuclear factor kappaB expression. A procaspase-9 construct with a point mutation that abrogates the release of the CARD abolished nuclear factor kappaB activation. Most importantly, the free CARD is shown to enhance the expression of the gene encoding the antiapoptotic Bcl-x protein and to strongly inhibit apoptosis. This is the first demonstration that different domains of the same caspase protein have proapoptotic and antiapoptotic effects and suggests that the relative effects of these domains are important in regulating the balance between death and survival.

Animals↗

PubMed2002

Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation-induced cell death.

Urocortin (Ucn), is a peptide related to hypothalamic corticotrophin-releasing factor (CRF) and binds with a high affinity to the CRF-R2 beta receptor which is expressed in the heart. Ucn promotes cardiac myocyte survival against hypoxia reoxygenation (HR) injury and this involves activation of the mitogen activated protein kinase pathway (MEK1/2 p42/44 MAPK). In this study we report that Ucn stimulates the phosphorylation of protein kinase B (PKB/Akt) via phosphatidylinositol (PI) 3-OH kinase (PI-3 kinase). To investigate the signalling pathways that mediate the anti-apoptotic and cell survival effect of Ucn in hypoxia reoxygenation (HR), gene based inhibitors of MEK1/2, PI-3 kinase and Akt were over-expressed in rat neonatal cardiac myocytes and cell survival effects against HR were assessed. The dominant negative mutants of the MEK1/2, PI-3 kinase and Akt inhibited Ucn mediated cardioprotection in HR and active PI-3 kinase was itself cardioprotective. In addition, chemical inhibitors of the PI-3 kinase pathway, wortmannin and LY294002 inhibit Ucn mediated cardioprotection in HR in both neonatal and adult cardiac myocytes. Hence the PI-3 kinase/Akt pathway is required in addition to MEK1/2 to mediate Ucn cardioprotection in HR. To our knowledge this is the first report of the activation of the PI-3 kinase/Akt pathway by a member of the CRF family of peptides.

Androstadienes↗

PubMed2002

Cardiotrophin-1 (CT-1) can protect the adult heart from injury when added both prior to ischaemia and at reperfusion.

OBJECTIVES: To determine whether the cytokine cardiotrophin-1 (CT-1) can protect the adult heart against ischaemia/reperfusion when added either prior to ischaemia or at reperfusion. BACKGROUND: CT-1 has previously been shown to protect cultured embryonic or neonatal cardiocytes from cell death. To assess the therapeutic potential of CT-1, it is necessary to determine whether this effect can be observed in adult cardiac cells both in culture and most importantly in the intact heart. METHODS: We examined the protective effect of CT-1 both in cultured adult rat cardiocytes and in the rat intact heart. In both cases, the cardiac cells were exposed to hypoxia/ischaemia followed by reoxygenation/reperfusion and CT-1 was administered either prior to hypoxia/ischaemia or at reoxygenation/reperfusion. RESULTS: CT-1 has a protective effect in reducing ischaemic damage in the intact heart ex vivo as assayed by infarct size to area at risk ratio (20% compared to 35%). Similar protective effects against cell death were noted in adult cells in vitro. Both in vitro and ex vivo CT-1 can exert a protective effect when added at the time of reoxygenation/reperfusion as well as prior to the hypoxic/ischaemic stimulus (cell death reduced from 50 to 20% in TUNEL assay, infarct size to zone at risk ratio reduced from 35 to 20%). These protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway. CONCLUSION: CT-1 can protect adult cardiac cells both in vitro and in vivo when added both prior to or after the hypoxic/ischaemic stimulus. The potential therapeutic benefit of CT-1 when added at the time of reperfusion following ischaemic damage is discussed.

Age Factors↗

PubMedDate not supplied

Activated STAT-1 pathway in the myocardium as a novel therapeutic target in ischaemia/reperfusion injury.

The signal transducers and activators of transcription (STAT) factors function as modulators of cytokine signaling and sensors responding to UV and ischaemic stress. Recently, we have reported a number of transcriptional mechanisms that induce expression of pro-apoptotic genes following ischaemia/reperfusion (I/R) in cardiac myocytes. For example, we have shown that STAT-1 plays a role in enhancing apoptotic cell death in cardiac myocytes exposed to I/R. In contrast to STAT-1, STAT-3, which is activated by the IL-6 cytokine family, has cardioprotective effects in cardiac myocytes exposed to I/R. The induction of apoptosis in cardiac myocytes by STAT-1 is dependent on STAT-1 phosphorylation on serine-727. Furthermore, we have demonstrated that the C-terminal transactivation domain of STAT-1 is required to mediate apoptotic cell death in cardiac myocytes exposed to I/R. Finally, cardiac myocytes isolated from mice expressing a truncated C-terminal STAT-1 were more sensitive to I/R-induced cell death. The isolated intact hearts from these animals exposed to I/R injury, had larger infarct size and a greater number of TUNEL-positive myocytes than control hearts. Hence, these studies demonstrate that activated STAT-1 is a pro-apoptotic factor in cardiac myocytes exposed to I/R, and is therefore a potential therapeutic target for the prevention of cell death following I/R injury.

Animals↗