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Title: Therapeutic regulation of cytoprotective pathways in the vascular endothelium
Author: Hamdulay, Shahir Sirajuddin
ISNI:       0000 0004 2699 0735
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Vascular injury and endothelial dysfunction are recognised features of atherosclerosis, transplant vasculopathy, SLE and rheumatoid. Heme oxygenase-1 (HO-1) and complement inhibitory proteins (CIP) decay-accelerating factor (DAF) limit vascular injury. There is considerable interest in therapeutic manipulation of cytoprotective genes and statins induce HO-1 and DAF expression, while rapamycin (R) enhances HO-1 and protects against post-transplant vasculopathy. A therapeutic regimen combining immunosuppression with vasculoprotection has the potential to prevent accelerated atherosclerosis in systemic inflammatory diseases. Based on previous data generated in the lab, my project explored the combination of atorvastatin and rapamycin, using DAF and HO-1 as target genes. A combination of AT 0.5μM and R 1μM led to a synergistic increase in EC DAF expression 24-72h post-treatment (>200%, p<0.05). In contrast, no change in CD59 was seen. In vivo, AT+R enhanced DAF expression in the murine aorta when compared to treatment with AT or R alone. The functional relevance of this synergy was revealed by enhanced protection against complement-mediated lysis, when compared to EC treated with R or AT (35% v 75% and 55% respectively, p<0.05). The role of DAF was confirmed by loss of protection in the presence of a DAF inhibitory mAb. Mechanistically, AT+R increased HO-1 expression and activity, while HO-1 inhibition with ZnPP attenuated synergy. As a consequence of HO-1-activation, AT+R increased intracellular ferritin, while Fe2+ chelation with DFO suggested depletion of intracellular Fe2+ is important for synergy. AT+R enhanced PKCα phosphorylation, while a DN-PKCα adenovirus and a PKCα inhibitory peptide abrogated DAF induction (AT + R 250% v DN-adv 95%, p<0.05). AT and R-induced p38 MAPK phosphorylation and inhibition attenuated DAF upregulation (AT+R 270% v 120%, p<0.05). Transcriptionally, DAF induction by AT+R required activation and binding of CREB to the DAF promoter. Synergistic upregulation of DAF expression was reproduced by simvastatin and rapamycin, while combinations of AT and cyclosporine or mycophenolate were ineffective. Treatment with rapamycin and atorvastatin synergistically enhances DAF expression and protection against complement-mediated injury. Rapamycin increases HO-1 activity and intracellular ferritin, and this combined with HMG-CoA reductase inhibition and activation of PKCα, p38 MAPK and CREB, results in optimal DAF induction. Thus, rapamycin and statin combination therapy may represent a means by which vascular endothelium can be therapeutically conditioned against complement-mediated injury and is worthy of further study in vascular diseases.
Supervisor: Mason, Justin ; Dorian, Haskard Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral