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Biochemical mechanisms of endothelial and smooth muscle cell apoptosis induced by photodynamic therapy Granville, David James


Photodynamic therapy (PDT) is a clinically approved therapeutic modality for several types of cancer and age-related macular degeneration. PDT is under investigation for the treatment of cardiovascular conditions such as restenosis, transplant rejection and atherosclerosis. Although the vasculature is a major target of PDT in the treatment of these ailments, the cellular and biochemical effects of PDT on endothelial cells (EC) and smooth muscle cells (SMC) are largely unknown. Thus, my thesis work was focused on defining the biochemical processes underlying EC and SMC apoptosis in response to PDT. We hypothesized that PDT acts directly upon mitochondria to elicit rapid apoptotic cell death and involves the release of mitochondrial proteins and activation of caspases, and represents a balance of effects conveyed by members of the Bcl-2 family of proteins. PDT was shown to trigger mitochondrial release of cytochrome c and apoptosis, inducing factor (AIF), involving pro-apoptotic Bcl-2 homologues such as Bax and Bid, and activate caspases - 2,-3, -6, -7, -8 and -9 during EC and SMC apoptosis. Furthermore, AIF was shown to migrate from mitochondria to the nucleus, whereupon it is believed to initiate chromatin condensation and early (Stage I) DNA fragmentation. The effect of PDT on intracellular Ca²⁺ regulation in normal and Bcl-2-overexpressing cells was examined. Overexpression of the anti-apoptotic Bel- 2 proto-oncogene did not protect cells against PDT-mediated intracellular Ca²⁺ release. On the contrary, increased intracellular Ca²⁺ release was observed in Bcl-2 overexpressing cells. Additional studies showed that Bcl-2 overexpression was associated with increased endoplasmic reticular and mitochondrial Ca²⁺ load. PDT-induced Ca²⁺ release was due to the release of Ca²⁺ from E R and mitochondrial stores and was not due to an influx of intracellular Ca²⁺ from bathing culture media. Furthermore, extrusion of Ca²⁺ via plasma membrane Ca²⁺ ATPases was not hindered, while ER-mediated sequestration of Ca²⁺ was impaired. Impairment of ER-mediated sequestration of Ca²⁺ was shown to be due to caspase-independent degradation of sarco/endoplasmic reticulum Ca²⁺ ATPases (SERCA). In summary, our results offer key insights into the anti-tumour effects of PDT as well as its effects on EC and SMC. PDT was shown to induce rapid changes to both mitochondria and endoplasmic reticuli. Results pertaining to biochemical mechanisms of EC and SMC may eventually lead to the discovery of novel therapeutic modalities for the treatment of common vascular disorders.

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