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Title: Endothelial and myocyte apoptosis in the heart exposed to ischaemia/reperfusion injury
Author: Scarabelli, Tiziano Maria
ISNI:       0000 0001 3554 7717
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2003
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Apoptosis is a form of cell suicide, which contributes, together with necrosis, to the cardiac cell loss following ischaemia/reperfusion injury (IRI). This thesis focuses on the occurrence of apoptotic cell death in different cell types during ischaemia/reperfusion injury, the initiating pathways leading to activation of the apoptotic process as well as the clinical potential of new cardioprotective agents. In a first set of experiments, carried out in the isolated Langendorff perfused rat heart, we attempted to describe the occurrence of apoptosis in specific cell types during ischaemia- reperfusion injury, including the very early stages of reperfusion. Apoptosis was seen mainly in endothelial cells, only after the onset of reperfusion. In addition, using a three-step immunocytochemical technique, we showed that the first cells to appear as apoptotic in the very early stage of re perfusion were those in the coronary vasculature, followed in a later phase of reperfusion by cardiac myocytes. Furthermore, the number of positive myocytes decreased with increasing distance from the positive vessel, suggesting that cells of the coronary vasculature may actively contribute to the death of myocytes by releasing pro-apoptotic mediators. The apoptotic cascade can be initiated either by mitochondrial damage and activation of caspase-9, or by death receptor ligation, which leads to activation of caspase-8. In an other set of experiments, specific inhibitors of caspase-8 and caspase-9 were used in order to estimate the relative contribution of the two main apoptotic initiating pathways in inducing apoptosis both in endothelial cells and cardiac myocytes in the two phases of ischaemia reperfusion. Cleavage of caspase-9 was observed primarily in endothelial cells. Conversely, caspase-8 cleavage is only found in cardiomyocytes, where it progressively rises throughout reperfusion. Consistent with this finding, addition of a specific caspase-9 inhibitor to the perfusate before I prevented endothelial apoptosis, whilst pre-ischaemic infusion of a specific C8 inhibitor affected only myocyte apoptosis. Other experiments were aimed at studying the cardioprotective effects of minocycline in primary cultures of both neonatal and adult cardiac myocytes as well as in the intact heart. Minocycline is a second-generation tetracycline with proven safety that is used in humans for the treatment of acne and urethritis as well as of severe chronic inflammatory diseases. Our report shows that minocycline significantly reduces the post-ischaemic occurrence of necrotic and apoptotic cell death, with normalization of developed and diastolic pressure. In regard to its antiapoptotic mechanism of action, we observed that minocycline reduces the expression level of initiator caspases, increases the ratio of XIAP to Smac/DIABLO at both the mRNA and protein level, and prevents the mitochondria-mediated release of cytochrome c and Smac/DIABLO. These synergistic actions dramatically prevent the post-ischaemic induction of caspase activity associated with cardiac l/R injury. Owing to its safety record and multiple novel mechanisms of action, minocycline may be a valuable cardioprotective agent to ameliorate the cardiac dysfunction and cell loss associated with l/R injury We also investigated the hemodynamic, bioenergetic and cytoprotective effects of Urocortin, a 40 amino acid member of the Corticotropin Releasing Hormone family, highly expressed in the cardiovascular system. This endogenous cardiac peptide was administered ex vivo pre-l, pre-l and during R, and during R only, to isolated perfused rat hearts exposed to l/R. Significant, and in some treatment groups, complete recovery of end diastolic pressure and developed pressure was observed, together with reduction in endothelial and myocyte cell death. In the groups receiving Ucn pre-l, significant recovery of high-energy phosphate reservoirs was also seen. Since the cytoprotective and functional benefits are still produced when Ucn is given only at R, our data suggest that Ucn may be useful clinically in the management of myocardial infarction. During cardiopulmonary bypass, the cardioplegic arrest and subsequent reperfusion inevitably expose the heart to an iatrogenic ischaemia/reperfusion injury. This background encouraged us to evaluate the occurrence of apoptosis and the relative contribution of its signalling pathways in human myocytes from patients exposed to cardiopulmonary bypass, warm blood cardioplegia, and subsequent reperfusion. Furthermore, we investigated whether the above surgical IRI modifies the cardiac expression of urocortin as well as its potential involvement as a salvage mechanism. Our study shows for the first time that warm blood cardioplegia induces apoptotic cell death in cardiac myocytes. This myocyte apoptosis, which was shown to involve colocalisation between TUNEL and caspase-3 positive staining, appears to be mainly sustained by the mitochondrial caspase-9- mediated pathway. We demonstrated moreover that urocortin expression is increased only in those myocytes, which are not apoptotic, suggesting that endogenous urocortin can also protect the human myocardium from IRI.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available