Vascular reactivity to vasopressin and phenylephrine is potentiated by constrictor prostanoids (CP) in normotensive female (F) but not male (M) rat aorta and CP function is estrogen-dependent. This study investigated the effects of estrogen on CP function and arterial blood pressure (MAP) during development of aortic coarctation-induced hypertension (HT). M and F rats, (15-18 wks.) in four groups: normotensive (NT), hypertensive (HT), ovariectomized (OVX), and OVX estrogen-replaced (OE), underwent abdominal aortic coarctation or sham surgery (NT). At 14 days, SQ 29,548 (SQ, Thromboxane A2 (TXA2) receptor antagonist) was given i.v. to the groups. In another experiment, rats received Ridogrel (TXA2 receptor antagonist+TXA2 synthase (TXS) inhibitor) or vehicle (methyl cellulose) daily, for 14 days. Thoracic aortae were analyzed for morphology, incubated in Kreb’s Henseleit Buffer (KHB) ± angiotensin II (ANG II), or underwent continuous pulsatile flow and pressure experiments (PFP) with KHB ± ANG II. Perfusate was analyzed for thromboxane B2 (TXB2) and prostaglandin F1α (PGF1α). RT-PCR and immunohistochemistry were performed for TXS. MAP was higher in F-HT than in M-HT after 14 days. SQ infusion reduced MAP substantially more in F-HT and OE-HT than in others. Ridogrel prevented increases in MAP in F/OE-HT rats, but not M/OVX-HT. Basal release of TXB2 and PGF1α increased to a greater extent in F-HT than in M-HT relative to their controls. ANG II-stimulated TXB2 and PGF1α release increased to a greater extent in F-HT than in M-HT. With or without ANG II, TXB2 production in HT during PFP increased with estrogen. PGF1α increased during PFP with estrogen, however not with ANG II. Pressurization resulted in less diameter change in F and OE-HT than in OVX-HT. Elastin increased with HT (inhibited by Ridogrel) in all but M. Collagen increased in HT with estrogen (inhibited by Ridogrel). Neither OVX-HT nor Ridogrel had any effect on morphology. Estrogen increased TXS with HT. Estrogen enhanced vascular CP and MAP in F-HT by increased expression of TXS and collagen density in the vasculature indicating that in aortic coarctation-induced HT, CP are upregulated by estrogen. Specific forms of HT in human beings may involve estrogen-induced vascular CP upregulation.

Hypertension is a chronic medical condition, and a primary contributor to severe cardiovascular disease. It can increase mortality risk. Studies have found that a low-grade inflammation is involved in the development of hypertension. In addition, men have been found to have a higher risk of hypertension than age-matched women, while women have an increased risk of hypertension after menopause. Female sex hormones are considered to play a protective role in the development and progression of hypertension. Prostanoids are inflammatory mediators, and have important physiological roles on promoting or suppressing inflammation. Studying the combined roles of these two biochemical families in disease development will be helpful to understand the balance between a self-protective effect and inflammatory response in hypertension, and find effective biomarkers to predict the progression of hypertension. In my study, a reversed phase high performance liquid chromatography is used to develop the / Chemistry

In the human uterus prostaglandins (PG) PGE2, PGD2, PGI2, PGF2α and Thromboxane A2 (TXA2), also termed prostanoids, are synthesised and deactivated to 15-keto PGE2, J2 metabolites, 6-keto-PGF1α, 15-keto PGF2α and TXB2 respectively. However, not all metabolites have been analysed simultaneously within the same tissue. The primary objective of this thesis was to determine full uterine prostanoid profiles in human non-pregnancy, pregnancy and parturition, to better understand these processes and find suitable tocolytic targets. In addition, ten cytokines in human cervico-vaginal fluid (CVF) were measured according to interval to labour to test their suitability as labour onset predictors, with a view to developing a test to determine women at risk of preterm labour. Prostanoid analysis was carried out in endometrium (n=9) and myometrium (n=15- 16) donated by non-pregnant women and lower segment myometrium obtained from pregnant women (before (n=14) and after labour onset (n=7)) by liquid chromatography coupled with electrospray ionisation mass spectrometry (LC/ESIMS/ MS). Cytokines produced by CVF collected from pregnant donors (20-41 weeks gestation, n=2-10) were investigated using Enzyme-Linked Immunosorbent Assay (ELISA) or Luminex®. Human endometrium produced greater concentrations of TXB2, PGE2 and PGF2α than myometrium in vitro (p<0.05). Fifteen prostanoids were detected in human myometrium. Production of 6-keto-PGF1α, PGE1 and PGF1α increased whilst 15- keto PGE2 and PGJ2 decreased at term pregnancy (37-41 weeks gestation) versus non-pregnancy (p<0.05). Myometrium from parturient donors synthesised TXB2 and PGE2 more abundantly than the non-labouring equivalent. Cytokine concentration was greatest in CVF sampled the week before labour, in particular Interleukin-6 (IL-6), Macrophage Inflammatory Protein-1α (MIP-1α) and Monocyte Chemotactic Protein-1 (MCP-1) (p<0.05). Endometrial TXB2, PGE2 and PGF2α could aid in proliferation of glandular epithelium prior to ovulation. Prostacyclin may facilitate prolongation of pregnancy to term and thromboxane could contribute to uterine stimulation during labour. Cervical dilation may be influenced by PGE2 in lower segment myometrium. MCP- 1, MIP-1α and IL-6 could mark a short interval to labour onset.

A molécula CD14 é uma glicoproteína expressa na membrana de leucócitos e células não mieloides, e pode ser encontrada tanto na membrana plasmática ancorada por glicosilfosfatidilinositol (GPI), como na forma solúvel no espaço extracelular. Esta molécula atua como co-receptor associado a receptores semelhantes a Toll (TLR), dentre eles o TLR4, e auxilia na ativação celular após reconhecimento de padrões moleculares associados a patógenos (PAMPs). Macrófagos ativados por produtos microbianos, como pelo lipopolissacarídeo (LPS), liberam citocinas e óxido nítrico (NO) que participam do processo inflamatório e sinalizam para células vizinhas ou distantes sobre a presença de agente agressor ou alterações teciduais. Após a ativação dos macrófagos, também são produzidos eicosanoides (EICs) que são mediadores lipídicos, como as prostaglandinas (PGs), os tromboxanos (TXs) e os leucotrienos (LTs). Os EICs são derivados em parte dos corpúsculos lipídicos (CLs) que são organelas citoplasmáticas funcionais, dinâmicas, ricas em lipídios e enzimas. Resultados anteriores do nosso grupo já demonstraram o envolvimento da molécula CD14 na produção de mediadores lipídicos, sem, contudo, elucidar os mecanismos. Assim, empregando macrófagos derivados da medula óssea (MDMO) estimulados com alta (500 ng/mL) e baixa (10 ng/mL) concentrações de LPS, tivemos como objetivo investigar a participação do CD14 na formação de CLs e na produção de prostanoides. A relevância da molécula CD14 foi ainda investigada na ativação de MDMO pelo ácido araquidônico exógeno (AA) (estímulo não específico). Nossos resultados demonstraram que a molécula CD14 contribui não somente para a formação de CLs e produção de prostanoides, como PGE2 e TXB2, mas também é essencial para a síntese de mediadores inflamatórios, como NO, TNF-? e IL-10, principalmente quando estimulados com baixa concentração de LPS / The CD14 molecule is a membrane-bound glycoprotein expressed by leukocytes and nonmyeloid cells, which can be attached to the plasma membrane by glycosylphosphatidylinositol (GPI) or in its soluble form in the extracellular space. This molecule acts as a co-receptor associated with Toll-like receptors (TLRs), including TLR4, and assists in cell activation after recognition of molecular patterns associated with pathogens (PAMPs). Macrophages activated by microbial products, such as lipopolysaccharide (LPS), release cytokines and nitric oxide (NO) that participate in the inflammatory process and signal to nearby or distant cells about the presence of an aggressive agent or tissue changes. After activation of macrophages, the lipid mediators denominated eicosanoids, such as prostaglandins (PGs), thromboxanes (TXs) and leukotrienes (LTs) are also produced. Eicosanoids are derived in part from lipid droplets (LDs) which are functional, dynamic cytoplasmic organelles, rich in lipids and enzymes. Previous results from our group have already demonstrated the involvement of the CD14 molecule in the production of lipid mediators, without, however, elucidating the mechanisms. Thus, using bone marrow-derived macrophages (BMDM) stimulated with high (500 ng/mL) and low (10 ng/mL) LPS concentrations, we aimed to investigate the participation of CD14 in the formation of LDs and in the production of prostanoids. The relevance of the CD14 molecule was further investigated by the activation of BMDM by exogenous arachidonic acid (AA) (nonspecific stimulus). Our results demonstrated that the CD14 molecule contributes not only to the formation of CLs and the production of prostanoids, such as PGE2 and TXB2, but it is also essential for the synthesis of inflammatory mediators, such as NO, TNF-? and IL-10, especially when stimulated with low LPS concentration

Ao final da gestação de ratas se observa hiporeatividade a agentes constritores que tem sido atribuída à produção alterada de óxido nítrico (NO) e prostanóides. Nesse estudo foi observada a participação dos derivados da sintase de NO (NOS) e ciclooxigenase (COX) nas alterações de reatividade vascular à fenilefrina (FE) durante a gestação, bem como a interferência da tensão passiva inicial na importância relativa dessas enzimas. Foi avaliada a resposta vasoconstritora à fenilefrina em anéis de aorta de ratas não grávidas (NG, fase estro do ciclo estral) e grávidas (G, 19o e 20o dias de gestação) com o endotélio íntegro ou lesado, e após inibição da COX ou da NOS. Fixando a concentração de FE (10-7 M) e variando a tensão passiva inicial (0,25, 0,50, 0,75, 1 ou 2g) a que o anel foi submetido, foi observado aumento da magnitude da resposta vasoconstritora dependente do aumento do estiramento do tecido em G e NG, sendo anéis de aorta de G menos reativos à FE do que NG nas diferentes tensões. A retirada do endotélio, a inibição da NOS por L-NNA ou da COX por diclofenaco de sódio (DF) reverteram parcialmente essa hiporeatividade. A adição de concentrações cumulativas de FE em anéis de aorta de G e NG submetidos às tensões passivas de 0,5 e 1 g demonstrou, novamente, hiporeatividade associada à gestação. A retirada do endotélio foi capaz de igualar a resposta constritora à FE de G e NG. A inibição da via NOS/guanilato ciclase com L-NNA e ODQ aumentou a reatividade de G e NG não alterando as diferenças de ambos; no entando, utilizando 0,5 g de tensão passiva, a adição de L-NNA igualou a resposta constritora à FE de G e NG. A adição de DF diminuiu a reatividade de NG igualando os grupos nas tensões de 0,5 e 1,0 g. O relaxamento induzido por acetilcolina em vasos pré-contraídos com FE não foi alterado pela gestação. Os resultados sugerem que a hiporeatividade à fenilefrina em anéis de aorta de ratas ao término da gestação está relacionada a uma alteração da ação de fatores derivados do endotélio, da NOS e da COX, sendo que as condições iniciais determinam a importância relativa desses agentes. / Late pregnancy is associated with diminished reactivity to vasoconstrictor agents that may be mediated by altered production/release of nitric oxide (NO) and prostaglandins. In the present study the role of NO synthase (NOS) and cyclooxygenase (COX) derived products in the altered vascular reactivity to phenylephrine (PE) was investigated in the isolated aorta of late-pregnant rats at different basal conditions. The vasoconstrictor response to PE was recorded in aorta rings of non-pergnant (NP, estrous) and late pregnant rats (P, 19- to 20 day) in the presence and absence of the endothelium, and with COX or NOS inhibition. The contractile response to PE (10-7 M) augmented with increasing resting tension from 0.25 to 2.0 g in both P and NP aorta; however, the responses in aortic rings from P were smaller at all passive tension employed. Endothelium removal or NOS inhibition with L-NNA or COX inhibition with sodium diclofenac (DF) attenuated but did not abolished the hyporesponsivity of P aorta. Concentration-response curves induced by PE were also attenuated in aortic rings of P compared with those of NP at both 0.5 and 1.0 g of resting tensions. Endothelium removal potentiated the constrictor response induced by PE in both P and NP aortas and abolished the difference between the groups. The NOS/guanilato cyclase pathway inhibition enhanced the PE-induced responses in P and NP but aortas of P maintained at 1.0 g of passive tension still respond less to PE; at 0.5 g of resting tension, however, L-NNA abolished the differences between P and NP. DF blunted PE-induced contraction only in NP aorta at both resting tensions and abolished the difference between P and NP aorta responses to PE. The relaxant responses of aortic rings to acetylcholine were not influenced by pregnancy. These results suggest that the refractoriness to PE-induced contraction observed in aortic rings of late-pregnant rats is related to altered production of NOS and COX endothelial factors. In addition, the basal conditions determine the relative contribution of these factors to the observed refractoriness to vasoconstrictor agent.

Ao final da gestação de ratas se observa hiporeatividade a agentes constritores que tem sido atribuída à produção alterada de óxido nítrico (NO) e prostanóides. Nesse estudo foi observada a participação dos derivados da sintase de NO (NOS) e ciclooxigenase (COX) nas alterações de reatividade vascular à fenilefrina (FE) durante a gestação, bem como a interferência da tensão passiva inicial na importância relativa dessas enzimas. Foi avaliada a resposta vasoconstritora à fenilefrina em anéis de aorta de ratas não grávidas (NG, fase estro do ciclo estral) e grávidas (G, 19o e 20o dias de gestação) com o endotélio íntegro ou lesado, e após inibição da COX ou da NOS. Fixando a concentração de FE (10-7 M) e variando a tensão passiva inicial (0,25, 0,50, 0,75, 1 ou 2g) a que o anel foi submetido, foi observado aumento da magnitude da resposta vasoconstritora dependente do aumento do estiramento do tecido em G e NG, sendo anéis de aorta de G menos reativos à FE do que NG nas diferentes tensões. A retirada do endotélio, a inibição da NOS por L-NNA ou da COX por diclofenaco de sódio (DF) reverteram parcialmente essa hiporeatividade. A adição de concentrações cumulativas de FE em anéis de aorta de G e NG submetidos às tensões passivas de 0,5 e 1 g demonstrou, novamente, hiporeatividade associada à gestação. A retirada do endotélio foi capaz de igualar a resposta constritora à FE de G e NG. A inibição da via NOS/guanilato ciclase com L-NNA e ODQ aumentou a reatividade de G e NG não alterando as diferenças de ambos; no entando, utilizando 0,5 g de tensão passiva, a adição de L-NNA igualou a resposta constritora à FE de G e NG. A adição de DF diminuiu a reatividade de NG igualando os grupos nas tensões de 0,5 e 1,0 g. O relaxamento induzido por acetilcolina em vasos pré-contraídos com FE não foi alterado pela gestação. Os resultados sugerem que a hiporeatividade à fenilefrina em anéis de aorta de ratas ao término da gestação está relacionada a uma alteração da ação de fatores derivados do endotélio, da NOS e da COX, sendo que as condições iniciais determinam a importância relativa desses agentes. / Late pregnancy is associated with diminished reactivity to vasoconstrictor agents that may be mediated by altered production/release of nitric oxide (NO) and prostaglandins. In the present study the role of NO synthase (NOS) and cyclooxygenase (COX) derived products in the altered vascular reactivity to phenylephrine (PE) was investigated in the isolated aorta of late-pregnant rats at different basal conditions. The vasoconstrictor response to PE was recorded in aorta rings of non-pergnant (NP, estrous) and late pregnant rats (P, 19- to 20 day) in the presence and absence of the endothelium, and with COX or NOS inhibition. The contractile response to PE (10-7 M) augmented with increasing resting tension from 0.25 to 2.0 g in both P and NP aorta; however, the responses in aortic rings from P were smaller at all passive tension employed. Endothelium removal or NOS inhibition with L-NNA or COX inhibition with sodium diclofenac (DF) attenuated but did not abolished the hyporesponsivity of P aorta. Concentration-response curves induced by PE were also attenuated in aortic rings of P compared with those of NP at both 0.5 and 1.0 g of resting tensions. Endothelium removal potentiated the constrictor response induced by PE in both P and NP aortas and abolished the difference between the groups. The NOS/guanilato cyclase pathway inhibition enhanced the PE-induced responses in P and NP but aortas of P maintained at 1.0 g of passive tension still respond less to PE; at 0.5 g of resting tension, however, L-NNA abolished the differences between P and NP. DF blunted PE-induced contraction only in NP aorta at both resting tensions and abolished the difference between P and NP aorta responses to PE. The relaxant responses of aortic rings to acetylcholine were not influenced by pregnancy. These results suggest that the refractoriness to PE-induced contraction observed in aortic rings of late-pregnant rats is related to altered production of NOS and COX endothelial factors. In addition, the basal conditions determine the relative contribution of these factors to the observed refractoriness to vasoconstrictor agent.

In the human uterus prostaglandins (PG) PGE2, PGD2, PGI2, PGF2¿ and Thromboxane A2 (TXA2), also termed prostanoids, are synthesised and deactivated to 15-keto PGE2, J2 metabolites, 6-keto-PGF1¿, 15-keto PGF2¿ and TXB2 respectively. However, not all metabolites have been analysed simultaneously within the same tissue. The primary objective of this thesis was to determine full uterine prostanoid profiles in human non-pregnancy, pregnancy and parturition, to better understand these processes and find suitable tocolytic targets. In addition, ten cytokines in human cervico-vaginal fluid (CVF) were measured according to interval to labour to test their suitability as labour onset predictors, with a view to developing a test to determine women at risk of preterm labour. Prostanoid analysis was carried out in endometrium (n=9) and myometrium (n=15- 16) donated by non-pregnant women and lower segment myometrium obtained from pregnant women (before (n=14) and after labour onset (n=7)) by liquid chromatography coupled with electrospray ionisation mass spectrometry (LC/ESIMS/ MS). Cytokines produced by CVF collected from pregnant donors (20-41 weeks gestation, n=2-10) were investigated using Enzyme-Linked Immunosorbent Assay (ELISA) or Luminex®. Human endometrium produced greater concentrations of TXB2, PGE2 and PGF2¿ than myometrium in vitro (p<0.05). Fifteen prostanoids were detected in human myometrium. Production of 6-keto-PGF1¿, PGE1 and PGF1¿ increased whilst 15- keto PGE2 and PGJ2 decreased at term pregnancy (37-41 weeks gestation) versus non-pregnancy (p<0.05). Myometrium from parturient donors synthesised TXB2 and PGE2 more abundantly than the non-labouring equivalent. Cytokine concentration was greatest in CVF sampled the week before labour, in particular Interleukin-6 (IL-6), Macrophage Inflammatory Protein-1¿ (MIP-1¿) and Monocyte Chemotactic Protein-1 (MCP-1) (p<0.05). Endometrial TXB2, PGE2 and PGF2¿ could aid in proliferation of glandular epithelium prior to ovulation. Prostacyclin may facilitate prolongation of pregnancy to term and thromboxane could contribute to uterine stimulation during labour. Cervical dilation may be influenced by PGE2 in lower segment myometrium. MCP- 1, MIP-1¿ and IL-6 could mark a short interval to labour onset. / Allergan Inc.

Pulmonary arterial hypertension (PAH) is a rare, devastating disease with no cure. Current treatment consists of a cocktail of vasodilators which relieve symptoms of PAH but do not treat the cause. Thus, there is a need for novel drugs that target the underlying pathological causes of PAH. PAH is a multi-factorial, but one key contributor is the pro-inflammatory cytokine IL-6 which stimulates pro-inflammatory and pro-angiogenic signalling mediated by the JAK/STAT pathway. One way in which IL-6 signalling via JAK/STAT is inhibited is via SOCS3 in a type of negative feedback loop whereby IL-6 induces transcription of SOCS3, which then attenuates further JAK/STAT signalling. SOCS3 can also be induced by cAMP. This is interesting as prostanoids, a type of drug used in the treatment of PAH due to its vasodilator effects and the only type to show any efficacy improving the life expectancy of PAH patients, acts by mobilising cAMP. Thus, prostanoid stimulation of cAMP could potentially limit IL-6 signalling via the induction of SOCS3. This is a novel mechanism of prostanoids which has not previously been considered. This study investigated the capability of prostanoids to limit the pro-inflammatory/pro-angiogenic effects of IL-6 that enable PAH to develop. Initial experiments confirmed that vascular endothelial cells responded to prostanoids which increased SOCS3 and limited IL-6 signalling activity. Further experiments utilising SOCS3 KO endothelial cell models demonstrated prostanoid inhibition of IL-6 signalling was due in part to SOCS3. In conclusion, this project has confirmed that prostanoids do limit the pro-inflammatory effects induced by IL-6 and that this is in part due to SOCS3. Although the exact mechanism is yet to be discovered, it will be beneficial in the treatment of PAH as it provides currently unexploited drug targets which can be considered for future PAH therapies. / British Heart Foundation

Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in significant alteration of concentrations of PGE2 at environmentally relevant concentrations.

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