Anastasis Stephanou

Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury.

Background—Apoptosis contributes to cell loss after ischemia/reperfusion injury in the heart. This study describes the time course and level of apoptosis in different cell types in the intact heart during ischemia/reperfusion injury. Methods and Results—Isolated Langendorff-perfused rat hearts were subjected to perfusion alone (control) or to 35 minutes of regional ischemia, either alone or followed by 5, 60, or 120 minutes of reperfusion. Sections were stained by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) and propidium iodide and with anti-von Willebrand factor, anti-desmin, or anti-active caspase 3 antibodies; they were then visualized by confocal microscopy. Sections were also examined by electron microscopy. No TUNEL-positive cells were seen in control hearts or hearts exposed to ischemia alone. Early in reperfusion, TUNEL staining was colocalized with endothelial cells from small coronary vessels. Endothelial apoptosis peaked at 1 hour of reperfusion and, at this time, there was clear perivascular localization of apoptotic cardiac myocytes, whose number was inversely proportional to their distance from a positive vessel. After 2 hours of reperfusion, apoptotic cardiac myocytes assumed a more homogeneous distribution. Active caspase 3 labeling was seen independent of DNA fragmentation during ischemia alone, but it colocalized with TUNEL staining over the 3 time points of reperfusion. Immunocytochemical findings were confirmed by electron microscopy and Western blotting. Conclusions—In the very early stages of reperfusion, apoptosis is first seen in the endothelial cells from small coronary vessels. The radial spread of apoptosis to surrounding cardiac myocytes suggests that reperfusion induces the release of soluble pro-apoptotic mediators from endothelial cells that promote myocyte apoptosis.

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.

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.

Expression and protective effects of urocortin in cardiac myocytes

Reverse transcription PCR showed that mRNA encoding the CRH-like molecule, urocortin, is expressed in a rat cardiac myocyte cell line and in primary cultures of cardiac myocytes. Identity of the amplified with the published sequence was established by restriction mapping and direct sequencing. Expression of urocortin mRNA was increased 12-18 h after thermal injury. Urocortin peptide protected cardiac myocytes from cell death induced by hypoxia. The data suggest that urocortin is an endogenous cardiac myocyte peptide which modulates the cellular response to stress.

Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis

We have previously demonstrated that STAT‐1 plays a critical role in promoting apoptotic cell death in cardiac myocytes following ischemia/reperfusion (I/R) injury. Epigallocatechin‐3‐gallate (EGCG), the major constituent of green tea, has recently been reported to inhibit STAT‐1 activity in noncardiac cells. In the present study, we have assessed the protective effects of EGCG and green tea extract (GTE) infusion on both cultures of cardiac myocytes and the isolated rat heart. EGCG reduced STAT‐1 phosphorylation and protected cardiac myocytes against I/R‐induced apoptotic cell death. Moreover, EGCG reduced the expression of a known STAT‐1 pro‐apoptotic target gene, Fas receptor. More interestingly, oral administration of GTE as well as EGCG infusion limited the extent of infarct size and attenuated the magnitude of myocyte apoptosis in the isolated rat heart exposed to I/R injury. This reduction cell death was associated with improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart. This is the first report to show that consumption of green tea is able to mediate cardioprotection and enhance cardiac function during I/R injury. Because GTE‐mediated cardioprotection is achieved, at least in part, through inhibition of STAT‐1 activity, we may postulate that a similar action can be implemented in the clinical setting to minimize STAT‐1 activation levels in patients with acute coronary artery disease (CAD).

STAT-1: a novel regulator of apoptosis

Summary.  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.

Signal Transducer and Activator of Transcription-1 and Heat Shock Factor-1 Interact and Activate the Transcription of the Hsp-70 and Hsp-90β Gene Promoters

We have previously demonstrated that interleukin-6 (IL-6) increases the levels of the heat shock protein 90 (Hsp-90) and activates the Hsp-90β promoter via the IL-6-activated transcription factors NF-IL6 and signal transducer and activator of transcription-3 (STAT-3). Here, we show that interferon-γ (IFN-γ) treatment increases the levels of Hsp-70 and Hsp-90 and also enhances the activity of the Hsp-70 and Hsp-90β promoters with these effects being dependent on activation of the STAT-1 transcription factor by IFN-γ. These effects were not seen in a STAT-1-deficient cell line, indicating that IFN-γ modulates Hsp induction via a STAT-1-dependent pathway. The effect of IFN-γ/STAT-1 was mediated via a short region of the Hsp-70/Hsp-90 promoters, which also mediates the effects of NF-IL6 and STAT-3 and can bind STAT-1. This region also contains a binding site for the stress-activated transcription factor HSF-1. We show that STAT-1 and HSF-1 interact with one another via a protein-protein interaction and produce a strong activation of transcription, which is in contrast to our previous finding that STAT-3 and HSF-1 antagonize one another. To our knowledge this is the first report of HSF-1 interacting directly via a protein-protein interaction with another transcription factor. Such protein-protein interactions and the binding of a number of different stress and cytokine-activated transcription factors to a short region of the Hsp-90 and Hsp-70 gene promoters are likely to play a very important role in Hsp gene activation by non-stressful stimuli and the integration of these responses with the stress response of these genes.

CRH-like peptides protect cardiac myocytes from lethal ischaemic injury.

Simulated ischaemia causes both necrotic and apoptotic death of primary cultures of neonatal rat cardiac myocytes. Simulated ischaemia is associated with increased expression of urocortin mRNA and with the release of urocortin peptide into the medium. Exogenous urocortin is more potent than corticotropin releasing hormone (CRH) in protecting cardiac myocytes from necrotic and apoptotic death induced by ischaemia, and the cardioprotective effects of ischaemia-preconditioned media are abrogated by antagonists to the CRH family of peptides. Simulated ischaemia increases cardiac myocyte expression of CCAAT enhancer binding (C/EBP) transcription factors, and of the p65 subunit of NFkappaB, and reporter activity of a construct incorporating a fragment of the urocortin promoter containing a C/EBP consensus site is also enhanced by simulated ischaemia. The data suggest that ischaemia, acting partly through increased expression of C/EBP transactivators, increases expression of urocortin mRNA, which is rapidly translated to the mature form. The mature peptide is rapidly released, and exerts autocrine/paracrine protective effects through the cardiac CRH-R2 receptor which preferentially binds urocortin.

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.

Role of STAT-1 and STAT-3 in ischaemia/reperfusion injury

Ischaemia/reperfusion (I/R) injury results in the death of irreplaceable cardiac myocytes by a programme cell death or apoptosis. The signal transducers and activators of transcription (STAT) factors function as modulators of cytokine signaling and sensors responding to cellular stress. Interestingly, many studies have demonstrated that although they have a similar structural organization, STAT‐1 and STAT‐3 have apposing effects on processes such as differentiation or apoptosis. For example, STAT‐1 has been shown to induced apoptosis, whilst STAT3 is able protect cardiac myocytes following ischaemia/reperfusion (I/R) injury. Many of the effects of STAT‐1 and STAT‐3 involve the direct binding to DNA and transcriptional activation of target genes. However, recent studies have shown that for STAT‐1 some of its effects appear not to require DNA binding. For example, induction of apoptosis by STAT‐1 can be produced by the C‐terminal activation domain in the absence of the DNA binding domain. This therefore, appears to involve a co‐activator effect in which STAT‐1 is recruited to DNA via a DNA‐bound transcription factor. In this regard, it is of interest that STAT‐1 but not STAT‐3 has been shown to interact with p53 and enhance its growth arrest and apoptosis‐ inducing properties. Hence, the finding that STAT‐1 and STAT‐3 can modulate the apoptotic programme both by direct DNA binding or via a co‐activator mechanism and despite their very similar structures, suggests that these related factors may be therapeutic targets against the damage myocardium following I/R injury. Recently, we reported that the polyphenolic agent epigallocatechin‐3‐gallate (EGCG), a major constituent of green tea and a potent inhibitor of STAT‐1 activation, protects the myocardium against I/R injury.