Enhanced PGI2 Production Research Articles

Pharmacological Characterization of the Microsomal Prostaglandin E2 Synthase-1 Inhibitor AF3485 In Vitro and In Vivo.

RationaleThe development of inhibitors of microsomal prostaglandin (PG)E2 synthase-1 (mPGES-1) was driven by the promise of attaining antiinflammatory agents with a safe cardiovascular profile because of the possible diversion of the accumulated substrate, PGH2, towards prostacyclin (PGI2).ObjectivesWe studied the effect of the human mPGES-1 inhibitor, AF3485 (a benzamide derivative) on prostanoid biosynthesis in human whole blood in vitro. To characterize possible off-target effects of the compound, we evaluated: i)the impact of its administration on the systemic biosynthesis of prostanoids in a model of complete Freund's adjuvant (CFA)-induced monoarthritis in rats; ii) the effects on cyclooxygenase (COX)-2 expression and the biosynthesis of prostanoids in human monocytes and human umbilical vein endothelial cells (HUVECs) in vitro.MethodsProstanoids were assessed in different cellular models by immunoassays. The effect of the administration of AF3485 (30 and 100 mg/kg,i.p.) or celecoxib (20mg/kg, i.p.), for 3 days, on the urinary levels of enzymatic metabolites of prostanoids, PGE-M, PGI-M, and TX-M were assessed by LC-MS.ResultsIn LPS-stimulated whole blood, AF3485 inhibited PGE2 biosynthesis, in a concentration-dependent fashion. At 100μM, PGE2 levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB2 inhibition (40.56 ± 5.77%). AF3485 administration to CFA-treated rats significantly reduced PGE-M (P < 0.01) and TX-M (P < 0.05) similar to the selective COX-2 inhibitor, celecoxib. In contrast, AF3485 induced a significant (P < 0.05) increase of urinary PGI-M while it was reduced by celecoxib. In LPS-stimulated human monocytes, AF3485 inhibited PGE2 biosynthesis with an IC50 value of 3.03 µM (95% CI:0.5–8.75). At 1μM, AF3485 enhanced TXB2 while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB2. At 100 μM, maximal inhibition of the two prostanoids was associated with the downregulation of COX-2 protein by 86%. These effects did not involve AMPK pathway activation, IkB stabilization, or PPARγ activation. In HUVEC, AF3485 at 100 μM caused a significant (P < 0.05) induction of COX-2 protein associated with enhanced PGI2 production. These effects were reversed by the PPARγ antagonist GW9662.ConclusionsThe inhibitor of human mPGES-1 AF3485 is a novel antiinflammatory compound which can also modulate COX-2 induction by inflammatory stimuli. The compound also induces endothelial COX-2-dependent PGI2 production via PPARγ activation, both in vitro and in vivo, which might translate into a protective effect for the cardiovascular system.

Prostacyclin-synthase expression in head and neck carcinoma patients and its prognostic value in the response to radiotherapy.

Prostacyclin (PGI2 ) plays a role in cancer progression but the mechanism is currently poorly understood. Additionally, no data are available about the prognostic value of the PGI2 pathway in head and neck squamous cell carcinoma (HNSCC) therapy. We evaluated the expression of the PGI2 pathway in HNSCC patients. PGI2 production and PGI synthase (PGIS) expression, in terms of mRNA (RT-PCR) and protein (immunoblotting), were lower in tumour samples than in non-tumoural mucosa, whereas, as expected, COX-2 expression was increased in HNSCC tumour samples. Using local control of the tumour after radiotherapy or chemoradiotherapy as a dependent variable, patients were classified into two categories of PGIS transcript levels. The high-PGIS group had a significantly lower frequency of local and distant failure than the low-PGIS group, and the 5-year cancer-specific survival was higher [90.2% (95% CI 81.0-99.4%) versus 60.5% (95% CI 44.4-76.6%)]. None of the four HNSCC cell lines analysed expressed PGIS and therefore they did not produce PGI2 . However, HNSCC-conditioned media enhanced PGI2 production in endothelial cells (ECs). The stable analogue of PGI2 , carbaprostacyclin (cPGI2 ), exerted little effect on HNSCC cell line migration, and no effect on cell cycle distribution or proliferation rate after radiation injury was observed. Nevertheless, cPGI2 promoted EP-4-dependent in vitro angiogenesis. Von Willebrand factor expression (EC marker) and capillary density were significantly higher in the group of patients with high expression of PGIS. Our results indicate that PGIS expression was associated with radiotherapy efficiency. Although we do not provide direct evidence of a relationship between tumour vascularization and radiotherapy efficiency, our results suggest that the effect of PGI2 is related to its ability to promote vascularization. These results also support the concept that co-adjuvant therapy with PGIS enhancers, such as retinoids, could have therapeutic value for HNSCC treatment.

Biphasic regulation by dexamethasone of IL-1- and LPS-stimulated endothelial prostacyclin production.

Body reaction to injury comprises two major pathways: the immune response, predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in an increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied. Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10(-6) M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 micrograms/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI2 in a concentration- and time-dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24 + 2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below the baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase A2 (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulate during exposure to Dex.

Biphasic regulation by dexamethasone of IL-1-and LPS-stimulated endothelial prostacyclin production

Body reaction to injury comprises two major pathways: the immune response is predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied. Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10(-6) M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 microgram/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI2 in a concentration and time dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24 + 2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase A2 (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulate during exposure to Dex.

Effect of cyclic GMP and sulfhydryl on prostacyclin production by human vascular endothelial cells.

The relationship between sulfhydryls and cGMP has been observed in several biological processes. Captopril is a sulfhydryl-containing angiotensin converting enzyme (ACE) inhibitor, that decreases PGI2 production in cultured human vascular endothelial cells. Enalapril does not appear to have this property. The role of cyclic GMP (cGMP) and sulfhydryls in the regulatory mechanisms in captopril-induced PGI2 production and Ca++ kinetics was investigated. Bradykinin and Ca ionophore A23187 enhanced PGI2 production, and increased the cytosolic free Ca++ concentration ([Ca++]i). It was observed that 8-bromo cGMP increased intracellular cGMP concentration ([Ca++]i), and decreased PGI2 production without changing [Ca++]i. Sulfhydryl containing compounds such as captopril, N-acetylcysteine and glutathione decreased PGI2 production via increased [cGMP]i. Enalapril, an ACE inhibitor without sulfhydryls, has no effect on PGI2 production, [Ca++]i and [cGMP]i. These results suggested that the presence of sulfhydryl groups is an important factor in the ability of vasoactive substances to induce PGI2 production.

Enhancement of thrombin‐ and lonomycin‐stimulated prostacyclin and platelet‐activating factor production in cultured endothelial cells by a tumor‐promoting phorbol ester

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.

Vitamin E enhances prostacyclin production by cultured aortic endothelial cells.

The effect of vitamin E on prostacyclin (PGI2) production by cultured bovine aortic endothelial cells was evaluated in vitro. We found that human plasma-derived serum (PDS) conveys a stimulatory effect on PGI2 production by cultured vascular endothelial cells. In the present study, therefore, PDS was used as a physiological stimulant for PGI2 production. There was no effect of vitamin E alone on PGI2 production without stimulation by PDS. However, simultaneous addition of vitamin E and PDS to the cultures enhanced PGI2 production in a dose-dependent manner. Furthermore, preincubation with vitamin E prior to stimulation with PDS also enhanced PGI2 production. The enhancing effect of preincubation with vitamin E was demonstrated to occur in a time- and dose-dependent manner. These results clearly indicate that vitamin E at physiological doses can enhance PGI2 production by cultured bovine aortic endothelial cells stimulated with an agent such as PDS. It seems likely that vitamin E may contribute to increase PGI2 synthesis by the vascular wall in vivo, thereby reducing platelet aggregation and thrombus formation.

IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis.

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.

Additive effects of il-1 and tnf on induction of prostacyclin synthesis in human vascular endothelial cells

The effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on the induction of prostacyclin (PGI2) synthesis in human vascular endothelial cells (EC) was investigated. Both IL-1 and TNF increased PGI2 production by EC in both a time- and dose-dependent manner, and a combination of the two cytokines additively enhanced PGI2 production. Metabolic inhibitors including indomethacin and cycloheximide abolished cytokine induced PGI2 synthesis. Since, the effect of TNF was not inhibited by neutralization with antibody to IL-1 alpha and IL-1 beta, it seems that the mechanism is not mediated by endogenous IL-1 induced by TNF.

Thromboxane A2 and prostacyclin release in bleeding time blood during primary haemostasis in healthy individuals.

It is generally believed that prostacyclin (PGI2) generation is greatly stimulated when blood vessels are injured, even by minor trauma, such as venepuncture. The Simplate technique for measuring skin bleeding time was adapted to quantify by radioimmunoassay PGI2 and thromboxane A2 (TXA2) in the emerging blood, as the stable degradation products 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2), both of which were measured in venous plasma as well as in serum (clotted at 37 degrees C for 1 h). During bleeding, when platelets aggregate to occlude the injured vessels, the median TXB2 level in the emerging bleeding time blood was 1.7 ng/ml. The median TXB2 level in plasma was less than 1 ng/ml and in serum 275 ng/ml. The levels of immunoreactive 6-keto-PGF1 alpha were always below determination limit in bleeding time blood (0.2 ng/ml) and in plasma (0.1 ng/ml), whereas in serum the levels ranged between 0.26 and 0.47 ng/ml. The fact that enhanced PGI2 production in primary haemostasis in skin incisions could not be demonstrated calls for further investigations of possible PGI2 production with more sensitive assays or in injured large vessels.

Prostacyclin production by endothelial cells. Effects of sera from normal and hyperlipidemic subjects.

The influence of hyperlipidemic sera on prostacyclin (PGI2) production by cultured endothelial cells was assessed by comparing sera from three types of hyperlipidemias with sera from normal subjects. Sera prepared from normal whole blood (WBS), platelet-rich plasma (PRPS), and platelet-poor plasma (PPPS) were also compared. Bovine aortic endothelial cells (BAEC) incubated with 25% WBS increased PGI2 synthesis significantly within 1 hour, with little further increase by 16 hours; human umbilical vein endothelial cells (HUEC) incubated with 25% WBS for 1 hour showed no elevation in PGI2, whereas PGI2 levels increased substantially after 16 hours. PPPS and PRPS stimulated PGI2 synthesis by BAEC equally at 1 hour. However, there was no rise in PGI2 after PPPS in HUEC; PGI2 rose after 16 hours with PRPS and rose further with WBS after 16 hours. Since WBS best enhanced PGI2 production in human endothelial cells, it was chosen for comparison of the effects of hyperlipidemic and normolipidemic sera. PGI2 synthesis by HUEC significantly increased upon incubation with WBS from Types IIb and IV patients in comparison to WBS from Type IIa hypercholesterolemic patients or normal controls. In contrast, WBS from all these hyperlipidemic subjects stimulated PGI2 synthesis by BAEC similarly to WBS from controls. We conclude that incubation of human endothelial cells with WBS containing high levels of atherogenic lipoproteins does not reduce PGI2 formation by the cells. Moreover, the time course and the contribution of lipid, plasma, or cellular factors to PGI2 formation vary according to the cell type tested. Caution should be exercised in extrapolating results achieved with serum and cells from the same species to other settings.