Cyclooxygenase (COX) is the rate limiting enzyme that catalyses the production of prostanoids, which are crucial to vascular homeostasis. Evidence suggests that endothelial dysfunction and inflammation play a role in vascular complications in aging and diabetes. Previous animal studies by our laboratory at RMIT University reported enhanced COX expression with aging in rat aortas, platelets and monocytes. Potentially, alteration in COX expression may result in an imbalanced prostanoid production favoring the synthesis of vasoconstrictors and hence increase the risk of cardiovascular events in the aging population. The regulation of altered COX expression in aging, however, is not clear. It has been suggested that histone hyperacetylation may be an important mechanism that regulates COX levels during the aging process as increased histone acetylation has been shown to occur with aging. Thus, we hypothesized that COX expression is modulated by histone hyperacetylati on. This was investigated by measuring COX expression in histone hyperacetylated cultured endothelial cells. In the case of diabetes, studies have reported that the development of diabetes and its complications is associated with persistent inflammatory activity, evident with increased inflammatory markers in the circulation. COX-mediated pathways may be involved in this inflammatory process in diabetes. Furthermore, the formation of advanced glycation end products (AGEs) is accelerated in diabetes. AGEs can bind to receptors for AGEs (RAGE), which has also been suggested to play a role in inflammation in diabetes. We hypothesized that COX- and RAGE-mediated pathways contribute to increased inflammation in diabetes and potentiate the development of diabetic vascular complications. This was investigated by measuring changes in COX-mediated pathways in both rat and human diabetic models. The current thesis reports: 1) in cultured endothelial cells, histone hyperacetylation was associated with increased COX expression; 2) an overall increase in inflammation was observed in diabetes involving COX- and RAGE-mediated pathways. This was supported by increased platelet COX-1 and monocyte COX-2 levels in Zucker rats, increased monocyte COX-2 in human Type 1 diabetes and elevated plasma TXB2 and PGE2 levels in both human Type 1 and Type 2 diabetic subjects. Up-regulation of RAGE expression was further found in platelets and monocytes in both human diabetes types. When treated with NSAIDs, plasma prostanoid levels, COX and RAGE expression were reduced significantly in both platelets and monocytes in human diabetic subjects. 3) It is unclear how COX and RAGE expression was regulated, but histone modifications may be one of the mechanisms. Data from cultured cells indicated that increased COX expression was associated with increased histone acetylation levels induced by TSA. Concurrent increases in histone acetylation and COX-2 levels were also observed in human Type 1 diabetes, but similar findings were not observed in human Type 2 diabetes. In addition, we failed to find an age-dependent increase in monocyte histone H4 acetylation in human Type 2 diabetes despite an age-dependent increase in monocyte COX-2 expression. Thus, whether histone hyperacetylation modulates COX expression and in what conditions require further investigation.

Pharmaceuticals and personal care products are a growing concern in the aquatic environment. Compounds from the class of non-steroidal anti-inflammatory drugs are commonly detected in surface waters and have the potential to negatively affect aquatic organisms. The purpose of this experiment was to determine the acute and chronic effects of ibuprofen on rainbow trout (Oncorhynchus mykiss). Cyclooxygenase (COX) activity, vitellogenin (VTG) concentration and ethoxyresorufin-O-deethylase (EROD) activity were evaluated following waterborne ibuprofen exposure of trout to 1 and 10 mg/L in the acute exposure and 1, 32 and 1000 μg/L in the chronic exposure, along with an experimental control, E2 control of 1000 μg/L and an E2-ibuprofen mixed treatment. Ibuprofen did not inhibit COX enzyme activity in either gill or kidney tissue. To evaluate the estrogenic effects of ibuprofen, VTG concentrations were measured; by the end of the 56 day chronic exposure VTG concentrations significantly increased in all of the ibuprofen treatments relative to the controls. EROD activity may have been inhibited by ibuprofen but definitive conclusions could not be made. These findings indicate that more research needs to be done studying ibuprofen in aquatic systems. / UOIT

No / There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.