Endothelial cell apoptosis is an important process during vasculature remodelling, angiogenesis and inflammation. Medical interest in endothelial cell apoptosis as a target for arthritis and solid tumour treatment has prompted biochemical and pharmacological investigation into the mechanisms controlling endothelial cell survival and angiogenesis. In recent years it has been hypothesised that control of endothelial cell apoptosis and the induction of angiogenesis may in part be due to the enzyme cyclooxygenase (COX)-2. COX-2 is involved in the metabolism of arachidonic acid to prostaglandins in mammals. This pathway has been implicated in controlling inflammation and angiogenesis through prostaglandin (PG) production and more recently has been shown to inhibit endothelial cell death. It was the aim of this study to investigate endothelial cell apoptosis and angiogenesis focussing on the role of COX-2, prostaglandins and endogenous apoptotic inhibitors in these pathways. Endothelial cell apoptosis was assessed by chromatin condensation, DNA fragmentation and caspase activation. Angiogenesis was investigated by examining capillary-like tubule formation. Endothelial cell apoptosis induction and angiogenesis inhibition was observed using the selective COX-2 inhibitor 5-bromo-2-(4-fluorophenyl)-3- (methylsulfonyl) thiophene (DuP-697) at a concentration 100 times lower than has previously been reported. Apoptosis was confirmed by induction of caspases 8, 9 and 3 over 8 hr and DNA fragmentation and condensation over 24 hr. The effects observed may be due to a selective inhibition of COX-2 as apoptosis induction and angiogenic inhibition only occurred when COX-2 was inhibited by selective and non-selective COX inhibitors. Induction of endothelial cell death was induced by treatment with two natural products that inhibit COX-2, namely curcumin and 6-shogaol (from turmeric and ginger respectively) although only at concentrations higher than were required to inhibit COX- 2. Both compounds induced chromatin condensation in endothelial cells and lurkat E6.1 cells with no DNA laddering or caspase induction. Further examination of the mechanisms of endothelial cell survival were investigated by assessing the endogenous expression of the apoptosis repressor with a caspase recognition domain (ARC) protein through examining reverse transcriptase CRT) - peR, native protein expression and transgenic over-expression in the endothelial cells. Endogenous expression of ARC was found in endothelial cells. However this expression declined during in vitro culture. Transgenic expression of ARC was found to increase levels of ARC in vitro. However it had no effect on apoptosis inhibition after 24 hr. The underlying mechanisms of cell death induction may be compound dependent in endothelial cells. Pharmacological inhibition of COX-2 and possibly PGE2 generation has detrimental effects on angiogenesis and endothelial cell survival. However inhibition of COX-2 by natural products at low concentrations may be advantageous in preventing tumour angiogenesis with no apoptosis induction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:427544 |
| Date | January 2005 |
| Creators | Churchman, Adrian Thomas |
| Publisher | University of Hertfordshire |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2299/14269 |
Page generated in 0.0023 seconds