• The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells.

      Urquhart, Paula; Parkin, Susan M.; Rogers, J.S.; Bosley, J.A.; Nicolaou, Anna (2002)
      The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated 14C-eicosanoids and (b) the incorporation of 14C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of 14C-prostaglandin F2a by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated 14C-prostaglandins was observed with the CLA mixture (IC50 100 ¿M). The cis9,trans11 and trans10,cis12 (50 ¿M) isomers individually inhibited the overall production of stimulated 14C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 ¿M), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of 14C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of 14C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of 14C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.
    • LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression.

      Kirkby, N.S.; Zaiss, A.K.; Urquhart, Paula; Jiao, J.; Austin, P.J.; Al-Yamani, M.; Lundberg, M.H.; MacKenzie, L.S.; Warner, T.D.; Nicolaou, Anna; et al. (2013-07-09)
      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.
    • PGE2 differentially regulates monocyte-derived dendritic cell cytokine responses depending on receptor usage (EP2/EP4).

      Poloso, N.J.; Urquhart, Paula; Nicolaou, Anna; Wang, J.; Woodward, D.F. (2013-07)
      Dendritic cells (DCs) are central players in coordinating immune responses, both innate and adaptive. While the role of lipid mediators in the immune response has been the subject of many investigations, the precise role of prostaglandins has often been plagued by contradictory studies. In this study, we examined the role of PGE2 on human DC function. Although studies have suggested that PGE2 specifically plays a role in DC motility and cytokine release profile, the precise receptor usage and signaling pathways involved remain unclear. In this report we found that irrespective of the human donor, monocyte-derived dendritic cells (MoDCs) express three of the four PGE2 receptor subtypes (EP2–4), although only EP2 and EP4 were active with respect to cytokine production. Using selective EP receptor antagonists and agonists, we demonstrate that PGE2 coordinates control of IL-23 release (a promoter of Th17, an autoimmune associated T cell subset) in a dose-dependent manner by differential use of EP2 and EP4 receptors in LPS-activated MoDCs. This is in contrast to IL-12, which is dose dependently inhibited by PGE2 through both receptor subtypes. Low concentrations (∼1–10 nM) of PGE2 promoted IL-23 production via EP4 receptors, while at higher (>50 nM), but still physiologically relevant concentrations, IL-23 is suppressed by an EP2 dependent mechanism. These results can be explained by differential regulation of the common subunit, IL-12p40, and IL-23p19, by EP2 and EP4. By these means, PGE2 can act as a regulatory switch of immune responses depending on its concentration in the microenvironment. In addition, we believe these results may also explain why seemingly conflicting biological functions assigned to PGE2 have been reported in the literature, as the concentration of ligand (PGE2) fundamentally alters the nature of the response. This finding also highlights the potential of designing therapeutics which differentially target these receptors.
    • Profile of eicosanoids produced by human saphenous vein endothelial cells and the effect of dietary fatty acids

      Urquhart, Paula; Parkin, Susan M.; Nicolaou, Anna (Harcourt, 2009-12-07)
      Human saphenous vein endothelial cells (HSVECs) derived from primary cultures of adult human veins constitute an excellent in vitro model for studying human endothelial metabolism. In this study we report the14C-labelled prostanoid profile of HSVECs under resting and stimulated conditions and the effect of the n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid on them. Results indicate that HSVECs while under resting conditions produce mainly prostaglandin F2 ¿(PGF2 ¿). After stimulation with calcium ionophore A23187, the cells were found to synthesise PGI2, PGE2and PGF2¿as major products and thromboxane B2and PGD2as minor products. Production of14C-labelled hydroxyeicosatetraenoic acids was not detected. Eicosapentaenoic acid was found to inhibit basal and stimulated prostanoid production whereas docosahexaenoic acid inhibited basal but strongly increased stimulated prostanoid production. These results may offer the basis for further studies aiming to investigate targets for pharmacological intervention in inflammatory conditions.