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Eicosanoids and vascular endothelial cell function
192
ErA & Eicosanoids 1997 - Edinburgh
Plenary Sessions Tuesday 22 July
T1
T2
Prostanoid Receptors - An Update Robert A Coleman: Pharmagene Laboratories Ltd, 2A Orchard Road, Royston, Herts, UK
EICOSANOIDS AND VASCULAR ENDOTHELIAL CELL FUNCTION Sei-itsu Murota, Tokyo Medical & Dental University, Graduate School, Tokyo, Japan
The classificationof prostanoid receptorsinto five basic types, DP, EP, FP, IP and TP, is now well accepted, as is the subdivision of EP-receptors into four types, EPI, EP2, EP3 and EP4. In addition, there is now strong additionalevidencefor subdivisionof DP, IP and possibly TP-receptors. A further level complexitywithin this family results from alternativesplicing of various of these receptor types, and in every case so far reported the differences are in the Cterminus region. Splice variance was first demonstratedfor EP3receptors, and it now appears that there are at least 10 naturallyoccurring variants across a range of species, at least six having been demonstratedin man. They are associatedwith different Gproteins and distinct signal transduction pathways, they are also differentially subject to down-regulation. More recently, splice variance has also been demonstratedin EP~, FP and TP-receptors. The discovery of a family of prostanoids which are not cyclooxygenase products, the isoprostanes,has raised the possibilityof an extension to the prostanoid receptor family. Although most evidence pointsto isoprostanesacting through 'classical' prostanoid receptors,some recent evidencesuggeststhat distinct isoprostanesensitive receptors may exist. Finally, there are some actions of prostanoids that resutt from an action at a nuclear receptor (PPAR~), and it has been suggestedthat these receptorsshould be included in the prostanoid receptor family. However, both the affinity and specificity of binding of these receptorsfor prostanoids are low, and it is questionablewhether such receptorsplay any role in physiologically-relevantactionsof prostanoids.
T3 Prostaglandins as m o d u l a t o r s of l y m p h o c y t e mediated inflammatory and humoral responses. Richard P. Phipps, Josue Padilla, Beth Graf and Sarah Harris. Cancer Center, University of Rochester School of Medicine, Rochester, New York, 14642, USA.
A recently emergent concept is that PGE2 can shape an i m m u n e r e s p o n s e and p r o m o t e antibody driven responses at the expense of an inflammatory response. Two classes of CD4 + T helper (Th) lymphocytes are defined based on secretion of distinct cytokine profiles. Thl cells secrete IL-2 and IFNy and promote inflammation, whereas the Th2 subset produces IL-4 and IL-10 and stimulates antibody responses, including I g E PGE2 blunts the production of the Thl cytokines 11-2 and IFNy, in contrast to IL-4 and IL-10 produced by Th2 cells. Antigen presenting macrophages may initially activate Th cells. Macrophages synthesize IL-12, a cytokine necessary for induction of Thl lymphocytes. However, in the p r e s e n c e of PGE2 macrophages downregulate their production of IL-12. Thus PGE2, via reducing macrophage-derived IL-12, and T h l derived IL-2 and IFNy, tips the immunological balance towards a Th2 response. In support of this concept, PGE2 enhances, up to 26-fold, IgE antibody production by B lymphocytes stimulated with Th2-derived IL-4. These observations indicate that early in an immune response high levels of PGE2 can promote a Th2-driven antibody (IgE) response at the expense of the T h l inflammatory response. In support of this hypothesis, patients with HIV, Hyper-lgE Syndrome, burn or trauma, produce high PGE2 levels concomitant with depressed IL-2 ( T h l ) levels and high Th2 responses, characterized by elevated IgE antibody levels. Thus PGE2 or specific PGE2 inhibitors may be useful as approaches to manipulate the immune response.
Endothelial cells are multi-functional cells producing many kinds of bioactive substances. When serially cultivated, HUVEC ( human umbilical vein endothelial cells) showed limited in vitro life spans of approximately 60-68 PDL (population doubling levels). In the cultured HUVEC, NO (nitric oxide) production and the producing ratio of PGIJTXA2 declined, while endothelin production increased with in vitro aging, suggesting that anti-thrombotic and antivasoconstrictive roles of endothelial cells may decrease with aging. The key enzyme of PGI 2 production is COX (cyclooxygenase). COX-1 is housekeeping enzyme, while COX-2 is inducible one. We prepared both COX-1 alone expressing cells and COX-2 alone expressing cells. Examination of the effect of NO on these COX activities showed that NO donor suppressed only COX-2 activity but not COX-I activity. Treatment of HUVEC with EPA (eicosapentaenoic acid, 3gg/ml) tor 48h caused increase in EPA content in the phospholipid fraction by 20 times when compared with the control cells. The EPA enriched HUVEC lost their angiogenic activity. To know the cause of this phenomenon, response of HUVEC to VEGF (vascular endothelial growth factor) was examined. To the normal HUVEC, VEGF caused dose dependent increase in the MAP kinase activity and the cell growth, however, in the EPA enriched HUVEC these effects of VEGF were significantly suppressed. Examination of the reason of the reduced VEGF effects showed the occurence of down-regulation of KDR/FIk-1 (one of the VEGF receptors) expression in the EPA enriched HUVEC.
ErA & Eicosanoids 1997 - Edinburgh
Plenary Sessions Tuesday 22 July
T1
T2
Prostanoid Receptors - An Update Robert A Coleman: Pharmagene Laboratories Ltd, 2A Orchard Road, Royston, Herts, UK
EICOSANOIDS AND VASCULAR ENDOTHELIAL CELL FUNCTION Sei-itsu Murota, Tokyo Medical & Dental University, Graduate School, Tokyo, Japan
The classificationof prostanoid receptorsinto five basic types, DP, EP, FP, IP and TP, is now well accepted, as is the subdivision of EP-receptors into four types, EPI, EP2, EP3 and EP4. In addition, there is now strong additionalevidencefor subdivisionof DP, IP and possibly TP-receptors. A further level complexitywithin this family results from alternativesplicing of various of these receptor types, and in every case so far reported the differences are in the Cterminus region. Splice variance was first demonstratedfor EP3receptors, and it now appears that there are at least 10 naturallyoccurring variants across a range of species, at least six having been demonstratedin man. They are associatedwith different Gproteins and distinct signal transduction pathways, they are also differentially subject to down-regulation. More recently, splice variance has also been demonstratedin EP~, FP and TP-receptors. The discovery of a family of prostanoids which are not cyclooxygenase products, the isoprostanes,has raised the possibilityof an extension to the prostanoid receptor family. Although most evidence pointsto isoprostanesacting through 'classical' prostanoid receptors,some recent evidencesuggeststhat distinct isoprostanesensitive receptors may exist. Finally, there are some actions of prostanoids that resutt from an action at a nuclear receptor (PPAR~), and it has been suggestedthat these receptorsshould be included in the prostanoid receptor family. However, both the affinity and specificity of binding of these receptorsfor prostanoids are low, and it is questionablewhether such receptorsplay any role in physiologically-relevantactionsof prostanoids.
T3 Prostaglandins as m o d u l a t o r s of l y m p h o c y t e mediated inflammatory and humoral responses. Richard P. Phipps, Josue Padilla, Beth Graf and Sarah Harris. Cancer Center, University of Rochester School of Medicine, Rochester, New York, 14642, USA.
A recently emergent concept is that PGE2 can shape an i m m u n e r e s p o n s e and p r o m o t e antibody driven responses at the expense of an inflammatory response. Two classes of CD4 + T helper (Th) lymphocytes are defined based on secretion of distinct cytokine profiles. Thl cells secrete IL-2 and IFNy and promote inflammation, whereas the Th2 subset produces IL-4 and IL-10 and stimulates antibody responses, including I g E PGE2 blunts the production of the Thl cytokines 11-2 and IFNy, in contrast to IL-4 and IL-10 produced by Th2 cells. Antigen presenting macrophages may initially activate Th cells. Macrophages synthesize IL-12, a cytokine necessary for induction of Thl lymphocytes. However, in the p r e s e n c e of PGE2 macrophages downregulate their production of IL-12. Thus PGE2, via reducing macrophage-derived IL-12, and T h l derived IL-2 and IFNy, tips the immunological balance towards a Th2 response. In support of this concept, PGE2 enhances, up to 26-fold, IgE antibody production by B lymphocytes stimulated with Th2-derived IL-4. These observations indicate that early in an immune response high levels of PGE2 can promote a Th2-driven antibody (IgE) response at the expense of the T h l inflammatory response. In support of this hypothesis, patients with HIV, Hyper-lgE Syndrome, burn or trauma, produce high PGE2 levels concomitant with depressed IL-2 ( T h l ) levels and high Th2 responses, characterized by elevated IgE antibody levels. Thus PGE2 or specific PGE2 inhibitors may be useful as approaches to manipulate the immune response.
Endothelial cells are multi-functional cells producing many kinds of bioactive substances. When serially cultivated, HUVEC ( human umbilical vein endothelial cells) showed limited in vitro life spans of approximately 60-68 PDL (population doubling levels). In the cultured HUVEC, NO (nitric oxide) production and the producing ratio of PGIJTXA2 declined, while endothelin production increased with in vitro aging, suggesting that anti-thrombotic and antivasoconstrictive roles of endothelial cells may decrease with aging. The key enzyme of PGI 2 production is COX (cyclooxygenase). COX-1 is housekeeping enzyme, while COX-2 is inducible one. We prepared both COX-1 alone expressing cells and COX-2 alone expressing cells. Examination of the effect of NO on these COX activities showed that NO donor suppressed only COX-2 activity but not COX-I activity. Treatment of HUVEC with EPA (eicosapentaenoic acid, 3gg/ml) tor 48h caused increase in EPA content in the phospholipid fraction by 20 times when compared with the control cells. The EPA enriched HUVEC lost their angiogenic activity. To know the cause of this phenomenon, response of HUVEC to VEGF (vascular endothelial growth factor) was examined. To the normal HUVEC, VEGF caused dose dependent increase in the MAP kinase activity and the cell growth, however, in the EPA enriched HUVEC these effects of VEGF were significantly suppressed. Examination of the reason of the reduced VEGF effects showed the occurence of down-regulation of KDR/FIk-1 (one of the VEGF receptors) expression in the EPA enriched HUVEC.