6-keto PGF1α Research Articles

Local intra-uterine Ang-(1-7) infusion attenuates PGE2 and 6-keto PGF1α in decidualized uterus of pseudopregnant rats.

BackgroundCyclooxygenase (COX)-derived prostanoids (PGE2, PGI2) are important contributors to the process of decidualization. Previous studies showed the presence of Ang-(1-7) in the primary and secondary decidualized zones of the implantation site at early pregnancy. Decreased concentrations of Ang-(1-7) were found in the decidualized uterus compared to the non-decidualized uterus of pseudopregnant rats, suggesting that low levels of Ang-(1-7) are required for successful decidualization at early pregnancy.MethodsTo understand the role of Ang-(1-7) in prostaglandin production in a decidualized uterus, induced by a bolus injection of sesame oil, Ang-(1-7) (24 μg/kg/h) or vehicle was then infused directly into the decidualized uterine horn using an osmotic minipump. The right horns were not injected or infused and served as non-decidualized uterine horns in both groups of animals.ResultsDecidualization increased PGE2 concentration in the uterus (0.53 ± 0.05 vs. 12.0 ± 3.2 pmol/mg protein, p < 0.001, non-decidualized vs. decidualized horns); Ang-(1-7) infusion attenuated the increase of PGE2 (12.0 ± 3.2 vs. 5.1 ± 1.3 pmol/mg protein, p < 0.01 control vs. Ang-(1-7) treated decidualized horns). The stable metabolite of PGI2 (6-keto PGF1α) was increased with decidualization (0.79 ± 0.17 vs. 3.5 ± 0.82 pmol/mg protein, p < 0.001, non-decidualized vs. decidualized horns). Ang-(1-7) infusion attenuated the increase in 6-keto PGF1α in the decidualized horn (3.5 ± 0.82 vs 1.8 ± 0.37 pmol/mg protein, p < 0.05 control vs. Ang-(1-7) treated decidualized horns). The circulating levels of 6-keto-PGF1a and TXB2 were decreased by Ang-(1-7) infusion, while no difference was observed in circulating PGE2. Although the global assessment of cleaved caspase 3 immunostaining, a marker of apoptosis, was unchanged within the Ang-(1-7) decidualized horn, there were localized decreases in cleaved caspase 3 staining in the luminal region in the decidualized uterus of Ang-(1-7)-treated rats.ConclusionsThese studies show that increased local uterine Ang-(1-7) alters the uterine prostaglandin environment, possibly leading to disruptions of early events of decidualization.

Endometrial and conceptus response to exogenous progesterone treatment in early pregnant gilts following hormonally-induced estrus

The aim of the study was to determine how supplementation with exogenous progesterone (P4) affects the expression of genes important for endometrial receptivity and conceptus development during the peri-implantation period in pigs. Gilts with PMSG/hCG-induced first estrus received daily injections of corn oil (CO; n=7) or P4 (n=7) on days 3 to 10 after insemination. The dose of P4 was 25mg/100kg BW on days 3 and 4 of gestation, then increased to 50mg/100kg BW on days 5 to 10. Blood samples were collected each day to monitor concentrations of circulating P4. Animals were slaughtered on day 12 of pregnancy to obtain endometrial tissue, conceptuses, and uterine luminal flushing (ULF). Gilts in the P4 group had consistently greater P4 concentrations in the blood compared with controls. Treatment of gilts with P4 increased uterine weight and enhanced total protein content and 6-keto PGF1α (a PGI2 metabolite) content in the uterine lumen. Moreover, injections of gilts with P4 increased the expression of prostaglandin endoperoxide synthase 2, microsomal PGE2 synthase and vascular endothelial growth factor A mRNA in the endometrium, but had no effect on gene expression in conceptuses. These results indicate that P4 supplementation stimulated the secretory activity of the endometrium and increased the expression of genes responsible for vascular function and luteotropic PGE2 synthesis, which are important components of successful pregnancy establishment in pigs. However, no effect of P4 treatment was detected in conceptuses.

PP.29.10] EFFECTS OF AMLODIPINE AND ATORVASTATIN ON ENDOTHELIAL VASOACTIVE MEDIATORS IN PATIENTS WITH METABOLIC SYNDROME

Objective: To study the effects of the amlodipine and atorvastatin on the blood levels of endothelial vasoactive mediators in patients with metabolic syndrome (MS).Design and method: In our study we included 60 patients with MS and mild arterial hypertension. Patients were randomized into three treatment groups: monotherapy with amlodipine, monotherapy with atorvastatin and a combination therapy with amlodipine and atorvastatin. Office blood pressure (BP), NO metabolite levels («Shimadzu», Japan), endothelin-1 (ET-1) («Biomedica Gruppe», Austria), thromboxane B2 (TxB2) and 6-keto-prostaglandin F1α (6-ketoPG F1.α) (Assay designs) were performed at baseline and after 24 weeks of the therapy. In the control group was had 20 healthy volunteers. Data were represented as median (Med) and 25%-75% interquartile range or mean and standard deviation. p-value < 0,05 was considered as statistically significant Results: Monotherapy with amlodipine and combination therapy with amlodipine and atorvastatin were associated with the significant decreased office BP. Initially TxB2, 6-ketoPG F1α, ET-1 levels were significantly higher at patients with MS then in control group. Difference in NO metabolite level didn’t reach statistical significance. Monotherapy with amlodipine and atorvastatin or in a combination showed significant improvements in several studied parameters of endothelial vasoactive mediators. The results are shown in table 1. Conclusions: Amlodipine and atorvastatin treatment was showed positive effects on the levels of vasoactive mediators thereby leading to the improvement of endothelial function in patients with MS.

A New Model to Study the Role of Arachidonic Acid in Colon Cancer Pathophysiology.

A significant increase in cyclooxygenase 2 (COX2) gene expression has been shown to promote cylcooxygenase-dependent colon cancer development. Controversy associated with the role of COX2 inhibitors indicates that additional work is needed to elucidate the effects of arachidonic acid (AA)-derived (cyclooxygenase and lipoxygenase) eicosanoids in cancer initiation, progression, and metastasis. We have recently developed a novel Fads1 knockout mouse model that allows for the investigation of AA-dependent eicosanoid deficiency without the complication of essential fatty acid deficiency. Interestingly, the survival rate of Fads1-null mice is severely compromised after 2 months on a semi-purified AA-free diet, which precludes long-term chemoprevention studies. Therefore, in this study, dietary AA levels were titrated to determine the minimal level required for survival, while maintaining a distinct AA-deficient phenotype. Null mice supplemented with AA (0.1%, 0.4%, 0.6%, 2.0%, w/w) in the diet exhibited a dose-dependent increase (P < 0.05) in AA, PGE2, 6-keto PGF1α, TXB2, and EdU-positive proliferative cells in the colon. In subsequent experiments, null mice supplemented with 0.6% AA diet were injected with a colon-specific carcinogen (azoxymethane) in order to assess cancer susceptibility. Null mice exhibited significantly (P < 0.05) reduced levels/multiplicity of aberrant crypt foci (ACF) as compared with wild-type sibling littermate control mice. These data indicate that (i) basal/minimal dietary AA supplementation (0.6%) expands the utility of the Fads1-null mouse model for long-term cancer prevention studies and (ii) that AA content in the colonic epithelium modulates colon cancer risk. Cancer Prev Res; 9(9); 750-7. ©2016 AACR.

Use of a balanced dual cyclooxygenase-1/2 and 5-lypoxygenase inhibitor in experimental colitis

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) play an important role in inflammatory bowel diseases (IBDs). We investigated the effects of flavocoxid, a dual COX/LOX inhibitor, in experimental colitis induced with either dinitrobenzenesulfonic acid (DNBS) or dextrane sulphate sodium (DSS)In the first model, colitis was induced in rats by a single intra-colonic instillation (25mg in 0.8ml 50% ethanol) of DNBS; after 24h animals were randomized to receive orally twice a day, flavocoxid (10mg/kg), zileuton (50mg/kg), or celecoxib (5mg/kg). Sham animals received 0.8ml of saline by a single intra-colonic instillation. Rats were killed 4 days after induction and samples were collected for analysis. In the second model, colitis was induced in rats by the administration of 8% DSS dissolved in drinking water; after 24h animals were randomized to the same above reported treatments. Sham animals received standard drinking water. Rats were killed 5 days after induction and samples were collected for analysis.Flavocoxid, zileuton and celecoxib improved weight loss, reduced colonic myeloperoxydase activity, macroscopic and microscopic damage, and TNF-α serum levels. Flavocoxid and celecoxib also reduced malondialdheyde, 6-keto PGF1α and PGE-2 levels while flavocoxid and zileuton decreased LTB-4 levels. In addition, flavocoxid treatment improved histological features and apoptosis as compared to zileuton and celecoxib; moreover only flavocoxid reduced TXB2, thus avoiding an imbalance in eicosanoids production.Our results show that flavocoxid has protective effect in IBDs and may represents a future safe treatment for inflammatory bowel diseases.

Time-dependent activation of prostacyclin and nitric oxide pathways during continuous i.v. infusion of serelaxin (recombinant human H2 relaxin).

In the RELAX-AHF trial, a 48 h i.v. serelaxin infusion reduced systemic vascular resistance in patients with acute heart failure. Consistent with preclinical studies, serelaxin augments endothelial vasodilator function in rat mesenteric arteries. Little is known about the contribution of endothelium-derived relaxing factors after a longer duration of continuous serelaxin treatment. Here we have assessed vascular reactivity and mechanistic pathways in mesenteric arteries and veins and the aorta after 48 or 72 h continuous i.v. infusion of serelaxin. Male rats were infused with either placebo or serelaxin (13.3 μg·kg(-1) ·h(-1) ) via the jugular vein using osmotic minipumps. Vascular function was assessed using wire myography. Changes in gene and protein expression and 6-keto PGF1α levels were determined by quantitative PCR, Western blot and ELISA respectively. Continuous i.v. serelaxin infusion augmented endothelium-dependent relaxation in arteries (mesenteric and aorta) but not in mesenteric veins. In mesenteric arteries, 48 h i.v. serelaxin infusion increased basal NOS activity, associated with increased endothelial NOS (eNOS) expression. Interestingly, phosphorylated-eNOS(Ser1177) , eNOS and basal NOS activity were reduced in mesenteric arteries following 72 h serelaxin treatment. At 72 h, serelaxin treatment improved bradykinin-mediated relaxation through COX2-derived PGI2 production. Continuous i.v. serelaxin infusion enhanced endothelial vasodilator function in arteries but not in veins. The underlying mediator at 48 h was NO but there was a transition to PGI2 by 72 h. Activation of the PGI2 -dependent pathway is key to the prolonged vascular response to serelaxin treatment.

PGE2 and 6-Keto-PGF1α Generation and Cyclic AMP in the Atria of Rats during Normoxia and Hypoxia

Circulating antibodies can be detected in chronic periodontitis patients given the presence of serum auto antibodies against β 1 -adrenoceptor (β1-AR) in periodontitis patients by using cardiac membranes or a synthetic β1-AR peptide corresponding to the second extracellular loop of human β 1 -AR as antigens. Periodontitis patients also exhibit enhanced atria contractility in normoxia and tonic contraction in hypoxia. Atenolol, synthetic β1 peptide and nifedipine abrogate the action of both Isoproterenol and β1 IgG on atria contractility in both experimental conditions. In turn, β 1 IgG display a partial agonist-like activity and modifies the contractility of isolated atria, accompanied by an accumulation of cyclic AMP nucleotide in normoxia and hypoxia. The autoantibody is able to provoke an increment in the concentrations of PGE2 and 6-ketoPGF 1α , which is abrogated by preincubating atria with adrenergic antagonist, synthetic β 1 peptide and prostanoid receptor antagonists. On this basis this study seeks to correlate the periodontitis infection to an increased risk of cardiac disease high lightening the role of β1 IgG in the alteration of myocardial contractility and the subsequent increment of prostaglandins (PGE 2 and 6-keto-PGF 1α ,) accompanied by an increment in the production of cyclic AMP, resulting in an effective adaptation of myocardium function in acute ischemia.

Ovarian stimulation with human chorionic gonadotropin and equine chorionic gonadotropin affects prostacyclin and its receptor expression in the porcine oviduct

Prostaglandins are well-known mediators of crucial events in the female reproductive tract, eg, early embryo development and implantation. Prostacyclin (PGI2) is the most synthesized prostaglandin in the human oviduct during the postovulatory period, indicating its important role in supporting and regulating the oviductal environment. The present study was undertaken to determine the influence of insemination and ovarian stimulation with human chorionic gonadotropin (hCG)/equine chorionic gonadotropin (eCG) on PGI2 synthesis in the porcine oviduct on day 3 post coitus. Mature gilts (n = 25) were assigned into 2 experiments. In experiment I, gilts were divided into cyclic (control; n = 5) and inseminated (control; n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5), and superovulated/inseminated (1,500 IU eCG, 1,000 IU hCG; n = 5) gilts. Parts of oviducts (isthmus and ampulla) were collected 3 days after phosphate-buffered saline treatment (cyclic gilts of experiment I) or insemination (all other groups). Expression of messenger RNA for PGI2 synthase (PGIS) and its receptor (IP) was measured by real-time reverse transcription polymerase chain reaction (real-time RT PCR) and protein levels using Western blots. Concentrations of the PGI2 metabolite 6-keto PGF1α were evaluated by enzyme immunoassay and localization of PGIS and IP in the oviductal tissues using immunohistochemical staining. Insemination by itself increased PGIS protein levels in the oviductal isthmus (P < 0.05) and IP protein expression in the ampulla (P < 0.05). The concentration of 6-keto PGF1α increased significantly in the oviductal ampulla after insemination (P < 0.05). Induction of ovulation decreased IP protein levels in the oviductal ampulla (P < 0.05), whereas superovulation reduced IP levels in both parts of the oviduct (P < 0.01). Synthesis of 6-keto PGF1α was reduced by induction of ovulation and by superovulation in the oviductal ampulla (P < 0.05). Immunohistochemical staining confirmed the presence of PGIS in the muscular layer of the isthmus and both mucosa and muscular layers of the ampulla. IP-positive cells were observed in both mucosal and muscular layers of the isthmus and ampulla. This study showed for the first time that PGI2 synthesis and IP expression are insemination dependent. Moreover, ovarian stimulation with hCG/eCG decreases IP expression and 6-keto PGF1α concentrations in porcine oviducts. Therefore, disturbances in PGI2/IP expression and synthesis may lead to disruption of the oviductal environment and, in turn, perturbed development of embryos and their transport to the uterus.

A theoretical model of biochemical control engineering based on the relation between oestrogens/progestagens and prostaglandins

A biological complex organism is involuntarily guided from all sides by measure and regulation systems. The human being is such a complex organism.Many cyclical processes are simultaneously at work, making it unclear how and why which process takes place at which moment. Noticeable examples are the 28-day menstrual cycle and the 40-week pregnancy. The time of activation in the middle of the menstrual is fairly clear. Hormonal changes also occur in this period. Why the hormonal changes occur, and what their relationship is with the activation of the processes is unclear. That is also the case during pregnancies. What is it that determines that a pregnancy should last an average of 40weeks? What causes the changes in a complicated pregnancy? What are those changes? Prostaglandin concentrations have been found to have some relationship with these changes, but the activation of these changes and how to examine them is unknown.Using an example from practical experience, this article illustrates what Horrobin and Manku already reported in 1977, namely, the properties of prostaglandin E1 and 6-keto pgF1α: reversal effect with elevated concentration. The properties described is exceptionally suitable for the time of activation in a biochemically regulated measure and regulation system.These properties can help explain the occurrence of physiological cycles. The known electronic saw-tooth wave has a biochemical analogue with this.This paper describes the presumed relationship between hormones and the accompanying prostaglandins with the hormone effects based on what is known regarding their concentrations progress. This relationship reveals the practical consequences of the experimentally found sensitivity of biochemical effects with regard to the accompanying prostaglandins.This paper shows how the theoretical relationship between effects of oestrogens and progestagens result in a curve that comprise observable aspects of the Basal Body Temperature Curve.The modulating and activating prostaglandins also affect local changes in blood circulation. These changes are visible on specific sites on the abdominal skin via viscerocutaneous reflex pathways.Changes in blood circulation at specific areas of the skin can be representative of pain. Pain that also frequently arises during activation processes.These changes can be seen and measured with non-contactual infrared thermography on the cutaneous surface, and moments of activation and pain can be determined.

Yerba mate (Ilex paraguariensis) improves microcirculation of volunteers with high blood viscosity: A randomized, double-blind, placebo-controlled trial

BackgroundElevated blood viscosity is a risk factor for atherosclerosis, thrombosis and other cardiovascular events. Our previous studies have suggested that consumption of Yerba mate tea (Ilex paraguariensis) has strong antioxidant and lipid-lowering properties in animals. The in vivo effects of Yerba mate on blood viscosity in humans, however, have not been studied. ObjectiveThis study aims to investigate the effect of Yerba mate tea on the reduction of blood viscosity and the improvement of microcirculatory parameters commonly regarded as risk factors for serious cardio and cerebrovascular disorders. Methods142 subjects with high blood viscosity were recruited in this randomized, double-blind, placebo-controlled study. Yerba mate tea or placebo (5g/day) was administered to different groups for 6weeks. After treatment, results of hemorheological indexes, nailfold microcirculation, 6-keto-PGF1α and TXB2 and lipid profiles of subjects in the Yerba mate tea group were compared with those in the placebo-receiving group. ResultsParameters of blood viscosity and microcirculation were improved in the subjects from the Yerba mate tea group but not in placebo-receiving patients. After treatment, whole blood viscosity, plasma viscosity and the Equation K value of erythrocyte sedimentation rate (ESRK) decreased significantly in the Yerba mate group. Meanwhile, shape, flow state and nailfold microcirculation appeared positively changed. Specifically, blood flow speeds accelerated gradually and nailfold weighted integral values decreased significantly. Moreover, the vasodilator 6-keto PGF1α increased while the thromboxane TXB2 decreased in serum samples of subjects in the Yerba mate-receiving group. Overall, Yerba mate tea-receiving subjects saw nearly all measured values improve to levels comparable to those characteristic of patients with normal microcirculation. ConclusionsThese results indicate the therapeutic capacity of Yerba mate tea in the treatment of high blood viscosity. Here, Yerba mate tea played a role in the regulation of various indexes of hemorheology, nailfold microcirculation, and the platelet aggregating factors 6-keto-PGF1a and TXB2. The regulation of these might be correlated with reduced blood viscosity and accelerating blood flow. Thus, Yerba mate tea may reduce some key risk-factors of cardiovascular disease. Daily consumption of Yerba mate tea may be a better-tolerated option for individuals with high blood viscosity and microcirculatory disturbance and as such, a novel preventative strategy for patients at-risk for vascular disease.

Nano-LC-MS/MS for the quantitation of prostanoids in immune cells.

Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F1α (6-keto PGF1α), prostaglandin F2α (PGF2α), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25pg/mL in the injected solution (75fg on column (o.c.)) for PGE2 and PGD2 and 37.5pg/mL (112.5fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4(+) and CD8(+) T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matterwhether they were obtained from sensitized mice or non-sensitized mice.

Effects Of A Selective Histamine H&lt;sub&gt;4&lt;/sub&gt;R Antagonist On Inflammation In A Model Of Carrageenan-Induced Pleurisy In The Rat

The histamine H₄ receptor (H₄R), recently cloned and identified, is a G-protein coupled histamine receptor family expressed in immune cells which plays an important role in inflammation. Recent data evidentiated that H₄R antagonists can decrease airway inflammation and hyperreactivity in animal models of asthma. In the present study we evaluated the effect of the selective H₄R antagonist JNJ7777120 (JNJ) in carrageenan-induced pleurisy, an in vivo model of inflammation, well characterized for cellular and molecular mechanisms. Intra-pleural administration of λ-carrageenan (1% w/v in 0.2 ml sterile saline) determined an intense recruitment of leucocytes in pleural exudates and in lung tissues, activated inducible nitric oxide (NO) synthase and cyclooxygenase-2, thus increasing the generation of harmful autacoids such as NO and pro-inflammatory prostaglandins, PgE₂ and 6-ketoPgF(1α), increased cellular and DNA oxidative stress, measured as malondialdehyde and 8-OH-deoxyguanosine and the local generation of IL-1β and TNF-α. Moreover, the activity of caspase-3, an early marker of apoptosis was also activated by λ-carrageenan injection. The pre-treatment with JNJ (5-10 mg Kg⁻¹ b.wt., given intrapleurally), 60 min before carrageenan markedly reduced all the studied parameters. This study clearly demonstrated that histamine H₄R antagonists have anti-inflammatory effects and could have potential therapeutic application for the treatment of inflammatory diseases.

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Study of molecular mechanism of Prostaglandin E1 in inhibiting coronary heart disease

Prostaglandin E1 has been used clinically for improving heart diseases. In this study, we examined the effect of Prostaglandin E1 on blood lipid levels, heart protein and genes expression in coronary heart disease (CHD) rats. Female rats were fed either a control diet or hypercholesterolemic diet for 14weeks. The feeding of a hypercholesterolemic diet (HCD) increased the serum TC, TG, and LDL-c levels, decreased the serum HDL-c, E2, P, FSH, LH and PRL levels in CHD rats. In addition, The feeding of a HCD diet markedly increased the content of serum TXA2, TXB2, and decreased the content of serum PGI2, and PGI2/TXA2, 6-Keto PGF1a. Furthermore, the feeding of a hypercholesterolemic diet markedly increased expression levels of myocardium Fas and Caspase-3 protein and mRNA levels, vascular endothelial growth factor and basic fibroblast growth factor mRNA, and decreased RyR2 mRNA in CHD rats. The feeding of Prostaglandin E1 for 14weeks significantly reversed these abnormal biochemical indexes in rats. These findings suggest that Prostaglandin E1 play a obvious heart protective effect. The mechanisms may be related to restraining the excessive activation of Fas and Caspase-3 protein and modulating some gene expressions associated with CHD.

Characterization of inflamin, the first member of a new family of snake venom proteins that induces inflammation

Unlike other sea snakes, Aipysurus eydouxii feeds exclusively on fish eggs. This unusual feeding habit prompted us to search for unique transcripts in their venom glands. In the present study we expressed a novel cysteine-rich secretory protein containing 94 amino acid residues that was identified in its cDNA library. As it induced inflammation and writhing in animals, this protein was named inflamin. It induced two waves of prostanoid production. The first wave peaked at 10 min and 6-oxo PGF1α (prostaglandin F1α) (6-keto PGF1α) was the major product. The second wave, specifically of 6-oxo PGF1α and PGE2 (prostanglandin E2), started after 2 h. In RAW 264.7 cells, COX-1 (cyclo-oxygenase-1) activity showed a transient increase at 10 min and is responsible for the first wave, but its expression was unaffected. COX-2 was induced after 3 h and is responsible for the second wave. Using specific inhibitors, we showed that cPLA2 (calcium-dependent phospholipase A2), and not sPLA2 (secretory phospholipase A2), iPLA2 (calcium-independent phospholipase A2) or DAG (diacylglycerol) lipase, plays a key role in arachidonate release. The cPLA2 activity showed a transient increase of 62% at 10 min; this increase was due to its phosphorylation and not due to an increase in its expression. Thus inflamin, the first member of a new family of snake venom proteins, leads to an increase in the cPLA2 and COX-1 activity resulting in inflammation and pain.

Human C-reactive protein induces endothelial dysfunction in biobreeding diabetic rats

C-reactive protein (CRP) levels in diabetes predict cardiovascular events. Also, human CRP (hCRP) exacerbated the proinflammatory, pro-oxidant and procoagulant states in a spontaneous model of type 1 diabetes mellitus (T1DM), the biobreeding (BB) rat. Since there is a paucity of data examining the role of CRP on endothelial dysfunction in animal models of diabetes, we tested this hypothesis in the diabetic BB rat. Diabetic BB rats (n = 4 per group) were injected with human serum albumin (HSA) or hCRP [hCRP = 20 mg/kg body weight; intraperitoneal (IP)] for three consecutive days. The rats were euthanized on day 4. Biomarkers that were assayed included endothelin-1 (ET-1), soluble intracellular adhesion molecule-1 (sICAM-1), Von Willebrand factor (vWF) and 6-keto prostaglandin F1-alpha (6-keto PGF1-α) in plasma. hCRP administration resulted in a significant increase in plasma levels. Furthermore, hCRP-treated rats had significantly increased circulating levels of ET-1 (1.12 ± 0.6 pg/mL versus 0.4 ± 0.21 pg/mL), vWF (45 ± 2.4 ng/mL versus 34 ± 7 ng/mL) and sICAM-1 (41 ± 3 ng/mL versus 34 ± 3.4 ng/mL) compared to HSA-treated rats (p < 0.05). There was no significant effect on 6-keto PGF1-α levels. Hence, in this preliminary report, we make the novel observation that hCRP induces endothelial dysfunction in a spontaneous model of T1DM, and this could have implications for the vascular complications in diabetics.

Abstract 643: Heterozygous Endothelial Endothelin-1 Overexpression Potentiates Endothelium-Dependent Contractions in the Carotid Arteries of Obese Mice

Elevated plasma levels of the vasoconstrictor peptide endothelin-1 (ET-1) are associated with cardiovascular risk factors such as obesity, diabetes and hypertension, in which endothelium-dependent contractions are prominent. Exogenous ET-1 promotes the release of endothelium-derived contracting factors (EDCF), but the role of endogenously produced ET-1 in these processes is unknown. Therefore, mice with tie-1 promoter driven endothelium-restricted heterozygous overexpression of ppET-1 (TET+/-) and WT littermates were kept on standard chow as lean controls or administered a high fat diet for 30 weeks to induce obesity. At sacrifice, fasting glucose levels were significantly elevated in obese animals (8.3±0.3 vs. 5.3±0.2 mmol/l in lean controls, n =6, P &lt;0.001). Isometric tension was measured in aortic and carotid arterial rings in wire myographs. In phenylephrine-contracted aortic rings, endothelium-dependent and −independent relaxations to acetylcholine and sodium nitroprusside, respectively, were unaltered between groups. In carotid arteries, the potency of phenylephrine to evoke contractions was greater in preparations from obese TET+/- mice ( pD 2 6.71±0.07 vs. lean TET+/- 6.34±0.13, n =5-6, P &lt;0.05), whereas there was no change in the contractile response to the α 1 -adrenergic agonist by diet-induced obesity in WT littermates. The augmented EDCF responses to acetylcholine of quiescent carotid arterial rings of obese animals were further potentiated by TET+/- ( E max 51.3±1.1% vs. 40.6±1.3% KCl in WT obese, n =6, P &lt;0.001). The production of 6-keto PGF 1α − the stable metabolite of prostacyclin − was increased significantly in preparations from obese TET+/- mice (238.4±30.0 vs. 127.0±12.7 pg/mL in obese WT littermates, n =4-6, P &lt;0.05). In the presence of L-NAME, TP receptor activation by U46619 was more effective in obese TET+/- ( E max 151.8±5.4% vs. 126.1±4.7% KCl in WT obese, n =4-6, P &lt;0.05). Overall, TET+/- had no effect on relaxations in obese animals, but contractile responses − EDCF-mediated ones in particular − were facilitated. The present results suggest that this is due to an increased production of vasoconstrictor prostanoids possibly combined with an augmented responsiveness of the underlying vascular smooth muscle.

The Lipid Mediator Protectin D1 Inhibits Influenza Virus Replication and Improves Severe Influenza

Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.

Inhibition of microsomal prostaglandin E synthase‐1 by aminothiazoles decreases prostaglandin E2synthesisin vitroand ameliorates experimental periodontitisin vivo

The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL-1β and a cell-free mPGES-1 activity assay, as well as on inflammation-induced bone resorption in vivo, using ligature-induced experimental periodontitis in Sprague-Dawley rats. Aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) and 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) reduced IL-1β-induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES-1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase-2. In ligature-induced experimental periodontitis, alveolar bone loss, assessed by X-ray imaging, was reduced by 46% by local treatment with TH-848, compared to vehicle, without any systemic effects on PGE2, 6-keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES-1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti-inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel-Lindberg, T. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo.

Assay Profiles of Prostanoids and Female Sex Hormones for Studying Potential Biomarkers of Hypertension

Aim: Prostanoids and female sex hormones play an important role in the progression of hypertension. However, their close-related structures make simultaneous separation and analyses difficult. A method was developed to separate and analyze seventeen important bioactive compounds including nine prostanoids and eight sex hormones in a short time. By applying the method to quantify prostanoids and female sex hormones in urine samples from hypertension patients, it becomes possible to find effective biomarkers for clinical diagnosis, prevention and treatment of hypertension. Materials and methods: A reversed phase high performance liquid chromatography is used to develop the separation method. A combination of up to nine prostanoids (PGD2, PGE2, 6-keto PGF1α, PGF2α, 11-dehydro TXB2, 8-iso PGF2α, 13,14-dihydro-15-keto PGA2, 13,14-dihydro-15-keto PGE2 and 15-deoxy Δ12,14 PGJ2) and eight female sex hormones (E1, E2, E3, progesterone, 2-OHE1, 4-OHE1, 16α-OHE1, and 2-MeOE1) are chosen as analytes. The separation is performed on Symmetry C18 4.6x250 mm column with 5 μm particle size. A UV detector is selected to determine the levels of analytes in a single 25-minute run. Results: The method is the first we are aware of successfully demonstrating the separation of the two different groups of bioactive compounds prostanoids and female sex hormones. It has been shown to have excellent selectivity, sensitivity, and accuracy over biologically relevant concentration ranges and has been tested on urine samples from hypertension patients.