PGI2 Formation Research Articles

Degree of Cyclooxygenase-2 Inhibition Modulates Blood Pressure Response to Celecoxib and Naproxen.

Non-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of adverse cardiovascular events via suppression of cyclooxygenase (COX)-2-derived prostacyclin (PGI2) formation in heart, vasculature, and kidney. The Prospective Randomized Evaluation of Celecoxib Integrated Safety versus Ibuprofen Or Naproxen (PRECISION) trial and other large clinical studies compared the cardiovascular risk of traditional NSAIDs (i.e. naproxen), which inhibit both COX isozymes, with NSAIDs selective for COX-2 (i.e. celecoxib). However, whether pharmacologically equipotent doses were used - that is, whether a similar degree of COX-2 inhibition was achieved - was not considered. We compared drug target inhibition and blood pressure response to celecoxib at the dose used by most patients in PRECISION with the lowest recommended naproxen dose for osteoarthritis, which is lower than the dose used in PRECISION. Sixteen healthy participants (19-61 years) were treated with celecoxib (100 mg every 12h), naproxen (250 mg every 12h), or placebo administered twice daily for seven days in a double-blind, crossover design randomized by order. On Day 7 when drug levels had reached steady state, the degree of COX inhibition was assessed ex vivo and in vivo. Ambulatory blood pressure was measured throughout the final 12h dosing interval. Both NSAIDs inhibited COX-2 activity relative to placebo, but naproxen inhibited COX-2 activity to a greater degree (62.9±21.7%) than celecoxib (35.7±25.2%; p<0.05). Similarly, naproxen treatment inhibited PGI2 formation in vivo (48.0±24.9%) to a greater degree than celecoxib (26.7±24.6%; p<0.05). Naproxen significantly increased blood pressure compared to celecoxib (differences in least-square means of mean arterial pressure: 2.5 mm Hg (95% CI: 1.5, 3.5); systolic blood pressure: 4.0 mm Hg (95% CI: 2.9, 5.1); diastolic blood pressure: 1.8 mm Hg (95% CI: 0.8, 2.8); p<0.05 for all). The difference in systolic blood pressure relative to placebo was associated with the degree of COX-2 inhibition (p<0.05). Celecoxib 200 mg/day inhibited COX-2 activity to a lesser degree than naproxen 500 mg/day, resulting in a less pronounced blood pressure increase. While the PRECISION trial concluded the non-inferiority of celecoxib regarding cardiovascular risk, this is based on a comparison of doses that are not equipotent.ClinicalTrials.gov identifier: NCT02502006 (https://clinicaltrials.gov/study/NCT02502006).

Aspirin modulates production of pro-inflammatory and pro-resolving mediators in endothelial cells.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.

COX-2, the dominant source of prostacyclin

In a recent issue of PNAS, Kirkby et al. (1) claimed that cyclooxygenase (COX)-1, not COX-2, is the dominant source of prostacyclin (PGI2). This conclusion was based on experiments using a flawed approach to estimating enzyme activity and PGI2 formation and a selective omission of data in the literature.

Impaired formation of vasodilators in peripheral tissue in essential hypertension is normalized by exercise training

This study examined vascular function and the adenosine system in skeletal muscle of patients diagnosed with essential hypertension (n = 10) and of normotensive (n = 11) patients, before and after aerobic training. : Before and after 8 weeks of aerobic training, the patients completed experiments in which leg blood flow was determined during infusion of adenosine, acetylcholine and during exercise (20 W); muscle interstitial fluid and femoral venous plasma were sampled via microdialysis probes during baseline conditions, exercise and adenosine infusion and resting muscle biopsies were obtained from muscle vastus lateralis. Before training, leg vascular conductance in response to arterial adenosine infusion was similar in the hypertensive and normotensive groups and the individual vascular response was positively correlated to that of both acetylcholine infusion (r = 0.66, P < 0.001) and exercise (r = 0.72, P < 0.001). Before training, interstitial adenosine concentrations during exercise and prostacyclin (PGI2) concentrations after adenosine infusion were lower in the hypertensive than the normotensive group (P < 0.05). In the hypertensive group, training did not affect the vasodilatory response to arterially infused adenosine but increased the formation of interstitial adenosine and PGI2 and lowered blood pressure. In the normotensive group, training resulted in lower (P < 0.05) leg vascular conductance in response to arterial adenosine infusion. The present data suggest that essential hypertension is associated with a reduced capacity to form adenosine and PGI2 at the skeletal muscle microcirculatory level, which is likely to contribute to the increased peripheral vascular resistance related to the disease. This impairment in vasodilator formation can be normalized by aerobic training.

Inhibitory effect of prostaglandin I2 on bone marrow kinetics of eosinophils in the guinea pig

Enhanced eosinophil trafficking from bone marrow to the tissue is a hallmark of allergic diseases. We have shown previously that PGI(2) markedly attenuates the locomotion of human eosinophils in vitro. Here, we set out to determine the effect of PGI(2) on the trafficking of bone marrow eosinophils in the guinea pig. Shape change of bone marrow eosinophils was determined by flow cytometry, and chemotaxis assays were performed using a transwell migration system. Eosinophil release from bone marrow of guinea pigs was investigated in the isolated, perfused hind-limb preparation. We found that PGI(2) prevented the mobilization of eosinophils from bone marrow and attenuated the shape change and chemotactic responses of bone marrow eosinophils. These effects were mimicked by iloprost and were prevented by the IP antagonist CAY10441 and the adenylyl cyclase inhibitor SQ22536. Immunohistochemical staining of bone marrow confirmed the expression of IPs by bone marrow eosinophils. The rate-limiting enzyme of PGI(2) formation, PGIS, was found in large mononuclear cells. These data show that IP activation negatively modulates the mobilization and locomotion of bone marrow eosinophils and might therefore also protect against exaggerated recruitment of eosinophils to inflammatory sites.

8-isoprostane F2α up-regulates the expression of type 5 phosphodiesterase in cavernosal vascular smooth muscle cells: inhibition with sildenafil, iloprost, nitric oxide and picotamide

To explore the possible role of of 8-isoprostane F(2α) (8-IPF(2α) ) in the aetiology of erectile dysfunction (ED), as the over-production of superoxide (O(2)(-)) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in the formation of 8-IPF(2α) in vascular tissue, which has similar properties to thromboxane A(2) (TXA(2) ). TXA(2) is vasoconstrictor and up-regulates the expression of NADPH oxidase and phosphodiesterase type 5 (PDE5). Cavernosal vascular smooth muscle cells (CVSMCs) were incubated with 8-IPF(2α) or the TXA(2) analogue, U46619, ±sildenafil, iloprost (a stable prostacyclin [PGI(2) ] analogue) or the nitric oxide (NO) donor NONOate for 16 h. The formation of O(2)(-) was then measured, PDE5 expression assessed using Western blotting and PGI(2) and 8-IPF(2α) formation measured using enzyme-linked immunoassays. 8-IPF(2α) promoted the formation of O(2)(-) , an effect inhibited by apocynin (an NADPH oxidase inhibitor) and up-regulated the expression of PDE5. Under identical incubation conditions, 8-IPF(2α) induced an increase in the formation of 8-IPF(2α) but reduced the formation of PGI(2) . All, these effects were reversed by sildenafil, iloprost, NONOate and picotamide. These data show that O(2) (-) derived from NADPH oxidase influences the relative balance of PGI(2) and 8-IPF(2α) in CVSMCs, which in turn alters the degree of PDE5 expression. This is a novel pathogenic mechanism underlying ED and a novel mechanism of action of sildenafil.

Expression of thromboxane synthase, prostacyclin synthase and thromboxane receptor in atherosclerotic lesions: Correlation with plaque composition

ObjectivesProstaglandins, such as thromboxane A2 (TxA2) and prostacyclin (PGI2), are bioactive lipid mediators that are implicated in the pathogenesis of atherosclerosis. In the current study, we tested the hypothesis that thromboxane synthase (TXAS), prostacyclin synthase (PGIS) and thromboxane receptor (TP) are expressed within the atherosclerotic lesion. MethodsAtherosclerotic aorta segments were obtained from low-density lipoprotein receptor deficient (LDL r-KO) mice on a high fat diet. Expression levels of TXAS, PGIS and TP were evaluated by real-time quantitative reverse transcription PCR, and immunohistochemistry; TxA2 and PGI2 biosynthesis was also assayed. ResultsAfter 8 weeks on the fat diet, aortic arches from LDL r-KO mice showed a significant increase in PGIS, TXAS, TP mRNA, TxA2 and PGI2 levels, when compared with controls. By contrast, after 16 weeks on the high fat diet PGIS and PGI2 were significantly reduced, whereas TXAS and TP message and protein and TxA2 levels were further and significantly increased in the atherosclerotic tissues when compared with the 8-week group. These changes correlated with the cellular composition of the atherosclerotic lesions. ConclusionsTXAS, PGIS and TP are all present within the atherosclerotic lesion areas, their levels change during progression of atherogenesis and contribute to TxA2 and PGI2 formation.

Prostanoids in health and disease

The prostanoids are a family of lipid mediators generated by the action of cyclooxygenase on a 20-carbon unsaturated fatty acid, arachidonic acid. Prostanoids are generated widely in response to diverse stimuli and, acting in a paracrine or autocrine manner, play important roles in normal physiology and disease. This review summarizes the current knowledge on prostanoid generation and the roles of individual mediators, their biosynthetic pathways, and their receptors in health and disease.

Microsomal prostaglandin E synthase‐1 inhibition in cardiovascular inflammatory disease

Prostaglandins (PGs), particularly PGE2 and prostacyclin (PGI2), are potent mediators of pain and inflammation. Both atherosclerosis and aortic aneurysm exhibit the hallmarks of inflammation. However, randomized trials of inhibitors of PG synthesis--nonsteroidal anti-inflammatory drugs--reveal that they predispose to cardiovascular risk. This appears to be consequent to inhibition of PGI2 and PGE2 formed by cyclooxygenase-2 (COX-2). Inhibitors of microsomal PGE synthase-1 (mPGES-1) are being developed for relief of pain and interest has focused on their potential impact on the cardiovascular system. Deletion of mPGES-1 retards atherogenesis and limits aortic aneurysm formation in hyperlipidaemic mice. However, it does not predispose to thrombogenesis and has a limited impact on blood pressure compared to inhibition of COX-2. This occurs despite the potential of the suppressed PGE2 in affording cardioprotection via its EP2 and EP4 receptors. However, deletion of mPGES-1 permits rediversion of the PGH2 substrate to other PG synthases and augmented formation of PGI2 and PGD2 mitigates this effect. However, increased PGI2 may also attenuate relief of pain. Pain relief seems likely to be a nuanced indication for mPGES-1 inhibitors, but they have therapeutic potential in syndromes of cardiovascular inflammation, cancer and perhaps in neurodegenerative disease. However, as the products of substrate rediversion vary according to cell type, these drugs may have contrasting impact amongst individuals at varied stages of disease evolution.

Transcriptional Inhibition of Protease-Activated Receptor-1 Expression by Prostacyclin in Human Vascular Smooth Muscle Cells

Stimulation of protease-activated receptor-1 (PAR-1) by thrombin causes vascular smooth muscle cell (SMC) mitogenesis and has been implicated in the vascular response to injury. Vascular injury is also associated with enhanced formation of PGE2 and PGI2 (prostacyclin). This study investigates whether PGI2 and PGE2 modify the expression of PAR-1 and the cellular response to thrombin in human SMC. The PGI2-mimetic iloprost (1 to 100 nmol/L) attenuated mRNA, total protein, and cell surface expression of PAR-1. This was associated with inhibition of thrombin-induced mitogenesis and migration. Comparable inhibition of PAR-1 expression was observed with the selective IP-receptor agonist cicaprost, the adenylyl cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine and the PKA activator dibutyryl-cAMP. Similar effects of PGE2 required micromolar concentrations. The specific PKA-inhibitor Myr-PKI prevented PAR-1 downregulation by iloprost. The potential role of Rho family GTPases in PAR-1 regulation was also investigated. Iloprost decreased Rac1 mRNA and the Rac1 inhibitor NSC23766 mimicked the inhibitory effects of iloprost on PAR-1 protein--but not mRNA. The Rho kinase inhibitor Y27632 did not influence PAR-1 expression. IP-receptor agonists may limit the mitogenic actions of thrombin in human SMC by downregulating PAR-1 via modulation of cAMP-/PKA- and Rac1-dependent signaling pathways.

Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis

Prostaglandin (PG) E(2) is formed from PGH(2) by a series of PGE synthase (PGES) enzymes. Microsomal PGES-1(-/-) (mPGES-1(-/-)) mice were crossed into low-density lipoprotein receptor knockout (LDLR(-/-)) mice to generate mPGES-1(-/-) LDLR(-/-)s. Urinary 11alpha-hydroxy-9, 15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M) was depressed by mPGES-1 deletion. Vascular mPGES-1 was augmented during atherogenesis in LDLR(-/-)s. Deletion of mPGES-1 reduced plaque burden in fat-fed LDLR(-/-)s but did not alter blood pressure. mPGES-1(-/-) LDLR(-/-) plaques were enriched with fibrillar collagens relative to LDLR(-/-), which also contained small and intermediate-sized collagens. Macrophage foam cells were depleted in mPGES-1(-/-) LDLR(-/-) lesions, whereas the total areas rich in vascular smooth muscle cell (VSMC) and matrix were unaltered. mPGES-1 deletion augmented expression of both prostacyclin (PGI(2)) and thromboxane (Tx) synthases in endothelial cells, and VSMCs expressing PGI synthase were enriched in mPGES-1(-/-) LDLR(-/-) lesions. Stimulation of mPGES-1(-/-) VSMC and macrophages with bacterial LPS increased PGI(2) and thromboxane A(2) to varied extents. Urinary PGE-M was depressed, whereas urinary 2,3-dinor 6-keto PGF(1alpha), but not 2,3-dinor-TxB(2), was increased in mPGES-1(-/-) LDLR(-/-)s. mPGES-1-derived PGE(2) accelerates atherogenesis in LDLR(-/-) mice. Disruption of this enzyme retards atherogenesis, without an attendant impact on blood pressure. This may reflect, in part, rediversion of accumulated PGH(2) to augment formation of PGI(2). Inhibitors of mPGES-1 may be less likely than those selective for cyclooxygenase 2 to result in cardiovascular complications because of a divergent impact on the biosynthesis of PGI(2).

Protease-activated receptors and endothelial prostanoid production

The principal mechanism through which serine proteases regulate cell behaviour is by activation of a unique family of G-protein coupled receptors, referred to as protease-activated receptors (PARs 1–4). The functional significance of PARs in endothelial cells is largely undefined and the intracellular consequences of their activation are poorly understood. We have shown that the serine protease thrombin, as well as PAR-1 and -2-selective peptides induces cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of PGI2, and this was inhibited by the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38mapk and pharmacological or molecular blockade of MEK or p38mapk strongly inhibited thrombin- and PAR-2-induced COX-2 expression and PGI2 formation. Thrombin, and peptide agonists of PAR-1 and PAR-2, increased luciferase activity in HUVEC infected with an NF-κB-dependent luciferase reporter adenovirus and this, as well as PAR-induced PGI2 synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IκBα. Thrombin- and PAR-2-induced COX-2 expression and 6-keto-PGF1α generation were markedly attenuated by the NF-κB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. PAR-1 or PAR-2 stimulation caused nuclear translocation and phosphorylation of p65-NF-κB, and thrombin-, but not PAR-2-induced p65-NF-κB phosphorylation was reduced by inhibition of MEK or p38mapk. Activation of PAR-4 increased phosphorylation of ERK1/2 and p38mapk without modifying NF-κB activation or COX-2 induction. These findings provide new insights into the molecular mechanisms used by proteases and PARs to regulate generation of the cytoprotective molecule PGI2 through changes in endothelial COX-2 expression.

Cyclooxygenase-2 Induction and Prostacyclin Release by Protease-activated Receptors in Endothelial Cells Require Cooperation between Mitogen-activated Protein Kinase and NF-κB Pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.

Inhibition of Prostacyclin Release by Cigarette Smoke Extract in Endothelial Cells is not Related to Enhanced Superoxide Generation and NADPH-Oxidase Activation

Exposure of human umbilical endothelial cells (ECs) to cigarette smoke extract (CSE) activated the NADPH-oxidase enzyme and increased the production of superoxide (O-2) as well as reactive oxygen species (ROS). CSE also inhibited the prostacyclin (PGI2) formation by ECs. Preincubation of ECs with diphenylene iodonium (DPI), the inhibitor of NADPH oxidase, blocked the increase of O-2 production, but neither lowered the ROS level nor prevented the inhibition of PGI2 formation in CSE-treated cells. Preincubation of ECs with a medium supplemented with 1 mM vitamin C did not decrease, but rather increased the O-2 production in CSE-treated cells. However, adding 1 mM glutathione (GSH) to vitamin C decreased the O-2 production, indicating that vitamin C was overwhelmed by the prooxidant in CS, and GSH enhanced the recycling process and spared vitamin C. The ROS level remained high in CSE-treated cells even after preincubation with vitamin C or vitamin C + GSH compared to the control cells. These results are discussed in light of the possible decrease of antioxidant enzyme activities in CSE-treated cells and the increase of cellular hydrogen peroxide (H2O2) generated from the CSE, which cause an imbalance between oxidizing species and the antioxidants producing oxidative stress in CSE-treated cells. These results demonstrate that CSE has a direct inhibitory effect on PGI2 formation and enhances the level of ROS in CSE-treated ECs, regardless of the activation of NADPH-oxidase.

Marked Interindividual Variability in the Response to Selective Inhibitors of Cyclooxygenase-2

Variability in response to drugs may influence both efficacy and safety. Cyclooxygenase (COX)-2 inhibitors pose a cardiovascular risk by potentially increasing the likelihood of thrombosis, hypertension, and atherogenesis. Differences between individuals in the response to COX-2 inhibitors would be expected to influence their susceptibility to cardiovascular complications. We examined the variability in degree and selectivity of COX-2 inhibition in humans in response to celecoxib and rofecoxib. Fifty healthy volunteers received placebo, rofecoxib (25 mg), and celecoxib (200 mg), randomized by order. COX-1 and COX-2 inhibition was determined using ex vivo and in vivo indices of enzymatic activity. A subset of 5 individuals underwent 5 replicate studies to estimate variability in drug response both within and between subjects. Despite the higher COX-2 selectivity of rofecoxib in vitro, the average selectivity attained by 25 mg rofecoxib and 200 mg celecoxib in vivo were not different. However, there was considerable variability at an individual level in the degree of COX-2 inhibition and selectivity attained by both drugs. Approximately one third of the variability was attributable to differences between individuals, suggesting the contribution of genetic sources of variance, such as candidate polymorphisms detected in COX-1 and CYP2C9. The actual degree of selectivity for inhibition of COX-2 achieved by the coxibs relates both to chemical properties of the drug and to factors within an individual that modulate drug response. These sources of variability might be exploited to identify patients uniquely susceptible to benefit or at developing risk of cardiovascular complications.

Cyclooxygenase-2-Derived Prostacyclin Confers Atheroprotection on Female Mice

Before menopause, women are relatively protected against cardiovascular disease. Although estrogen (E2) retards atherogenesis in animal models and improves endothelial function in women with hyperlipidemia, the mechanisms of atheroprotection remain to be defined completely. The prostaglandin PGI2 inhibits platelet activation, vascular smooth muscle contraction, leukocyte-endothelial cell interactions, and cholesteryl ester hydrolase. PGI2 analogs limit atherogenesis, and atherosclerotic lesions have a reduced ability to produce PGI2. Cyclooxygenase 2 (COX-2), expressed in vascular endothelial cells, catalyzes the formation of prostaglandin endoperoxide from arachidonic acid, and this subsequently is transformed to PgI2 in endothelial cells by the action of PGI 2 synthase. Selective COX-2 inhibitors retard the excretion of PGI2 metabolites. These mechanisms may be relevant to atheroprotection in women because E2 increases the expression of COX-2 in vascular tissues and promotes the in vitro production of PGI2. Male mice lacking low-density lipoprotein receptor (LDLR) developed atherosclerosis more rapidly than did females, despite the lack of differences in total plasma cholesterol or high-density lipoprotein cholesterol. Female mice lacking both PGI2 receptor and the LDLR developed more prominent aortic lesions than did LDLR-knockout females (ie, lacking the gene for the LDL receptor). This effect was not gene dose-dependent and was not apparent in male mice. Further observations indicated that PGI2 decreases the platelet activation that characterizes early atherogenesis in female mice. It appears that PGI2 serves as an antioxidant in female LDLR-knockout mice before atherogenesis and at its earliest stage. Adding hydrogen peroxide to cultured mouse aortic smooth muscle cells increased synthesis of PGI2 and lipid peroxidation. There was no change in the expression of COX-2. Peroxide increased reactive oxygen species, and this effect was augmented by the lack of PGI2 receptor expression. These observations suggest that the PGI2 receptor modulates oxidant stress under baseline conditions. Importantly, E2 stimulated COX-2 expression and PGI2 formation. These findings suggest that E2 receptor alpha-mediated COX-2 stimulation of PGI2 makes a substantial contribution to atheroprotection in female LDLR-knockout mice. It is possible that ongoing treatment of patients with selective COX-2 inhibitors might undermine protection from cardiovascular disease in premenopausal females.

Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

Prostacyclin (PGI(2)) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI(2) in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI(2) release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500 ng/ml-20 microg/ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3-9 h and caused a time- and dose-dependent release of 6-keto PGF(1 alpha), the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-alpha (TNF-alpha) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostaglandin 12 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6-keto PGF(1 alpha)-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA upregulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

Coxibs: can this class of drugs survive?

Coxibs: can this class of drugs survive?

Cyclooxygenase-2-derived endogenous prostacyclin enhances mouse embryo hatching

The role of prostaglandins (PGs) in embryo hatching remains controversial. In addition, there is no direct evidence that mouse embryos synthesize PGs. The effects of endogenous PG on mouse embryo hatching were evaluated by blocking endogenous PG synthesis with indomethacin. Specific cyclooxygenase (COX) inhibitors were used to identify the role of COX-1- and COX-2-derived PGs. An eicosanoid profile was generated by incubating blastocysts with [3H]arachidonic acid and analysing the metabolites by high performance liquid chromatography. The expression and the localization of COX-1, COX-2 and prostacyclin synthase (PGIS) were examined by western blot analysis and immunohistochemistry. The hatching of embryos cultured in 30 microl of protein-free medium was blocked by indomethacin (P = 0.007) or a selective COX-2 inhibitor (P = 0.004). Adding back iloprost, a prostacyclin analogue, abolished the effects of the COX-2 inhibitor. Prostacyclin was the most abundant PG produced by mouse blastocysts, which expressed COX-1, COX-2 and PGIS. COX-1, COX-2 and PGIS were expressed in 4-cell stage embryos and beyond; they were present in the inner cell mass and the trophectoderm of the blastocysts. Mouse embryos express COX-1, COX-2 and PGIS which catalyse the formation of PGI2; COX-2-derived PGI2 plays a critical role in embryo hatching.

Inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right-heart dysfunction, and refractory hypoxemia after cardiothoracic surgery

Inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right-heart dysfunction, and refractory hypoxemia after cardiothoracic surgery