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Title: In vitro and in vivo molecular analysis of cyclooxygenase-2 (COX-2)-dependent and COX-2 independent cytoprotective pathways in the vascular endothelium
Author: Alrashed, Fahad Abdulaziz M.
ISNI:       0000 0004 7656 9340
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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Purpose: Non-steroidal anti-inflammatory drugs (NSAIDs) are important for limiting pain and inflammation, although many increase the risk of cardiovascular events including myocardial infarction, stroke and heart failure. However, celecoxib (a selective COX-2 inhibitor), has been associated with a more positive cardiovascular profile. We explored the hypothesis that celecoxib induces anti-oxidant and anti-inflammatory genes in the vascular endothelium via AMP kinase (AMPK), thereby limiting inflammation-mediated injury. Methods/Results: In vitro cultured human umbilical vein endothelial cells were studied. Our results indicate that celecoxib induces cytoprotective genes; including heme oxygenase-1 (HO-1) and manganese superoxide dismutase (MnSOD). Importantly, these observations were reproduced in human aortic endothelial cells. In contrast, the traditional NSAIDs (ibuprofen and naproxen) failed to reproduce the cytoprotective actions of celecoxib. Subsequently, we found celecoxib increased expression of additional cytoprotective genes (Bcl-2, FHC, DAF and TXNRD1) in vitro, and importantly, celecoxib treatment of C57BL/6 mice led to increased HO-1 and ferritin heavy chain (FHC) expression in the aortic endothelium in vivo. The actions of celecoxib were shown to be independent of COX-2 by using dimethyl-celecoxib, which lacks COX-2 inhibitory properties and reproduced celecoxib's cytoprotective effects. Mechanistically, celecoxib led to the generation of mitochondrial reactive oxygen species, which activated AMPK leading to HO-1 and MnSOD induction. Celecoxib treatment led to AMPKα (Thr172) and CREB (Ser133) phosphorylation via a linear pathway, and the nuclear translocation of NRF-2. The effects of celecoxib were inhibited by siRNA-mediated depletion of AMPKα1, CREB-1 and NRF2. Further functional analyses revealed celecoxib to inhibit, via activation of AMPK, TNFα-mediated phosphorylation of NF-κB p65, p65 nuclear translocation, TNFα-induced VCAM-1 expression and IL-1β-induced IL-6 mRNA expression. Conclusion: Our data demonstrate that celecoxib regulates cytoprotective gene expression in ECs via a novel mtROS-AMPK-CREB-Nrf2-dependent pathway. A deeper understanding of the mechanisms of action of NSAIDs will inform clinical practice and ultimately assist the development of safer therapeutics.
Supervisor: Mason, Justin ; Angelini, Gianni ; Mitchell, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral