6-keto PGF1 Alpha Research Articles

Differentiation‐dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins

A thorough understanding of the mechanisms of adipocyte differentiation and metabolism is important for the prevention and/or treatment of obesity and its complications, including type 2 diabetes mellitus. A complex role for prostaglandins (PGs) in adipogenesis is suggested. We examined the expression and cellular localization of enzymes in the cyclooxygenase (COX) cascade that synthesize PGs as well as the PG profile as a function of differentiation status in 3T3-L1 cells. Murine 3T3-L1 preadipocytes were used as a model for studies of adipocyte differentiation induced by a hormone cocktail and compared with the parental fibroblastic line NIH 3T3. Both cell lines were incubated in maintenance medium or differentiation medium. Nine days after differentiation, the expression of enzymes in the COX cascade was evaluated by immunoblot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry, and PG formation was examined using enzyme immunoassay. A differentiation-dependent diminution of COX-1 and COX-2 mRNA and cognate proteins in 3T3-L1 cells was observed. PG release, including PGE(2), 6-keto PGF(1alpha), PGD(2) and 15d-PGJ(2), significantly decreased following differentiation in 3T3-L1 cells (anova/Tukey, p < 0.05). However, microsomal PGE synthase (mPGES) and lipocalin-type PGD synthase (L-PGDS) were selectively upregulated. Immunocytochemistry revealed that COX-1 and COX-2 became intracellularly more diffuse upon differentiation, whereas mPGES was redistributed to the nuclear compartment. Regulation of PG formation and COX-2 expression in 3T3-L1 cells is differentiation-dependent and involves changes in the levels of gene expression of the individual isoforms as well as redistribution of the enzymes within cellular compartments.

Vascular Endothelial Growth Factor (VEGF)-A165-induced Prostacyclin Synthesis Requires the Activation of VEGF Receptor-1 and -2 Heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(121) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(121), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.

The Unsaponifiable Fraction of Virgin Olive Oil in Chylomicrons from Men Improves the Balance between Vasoprotective and Prothrombotic Factors Released by Endothelial Cells

Minor components of virgin olive oil (VOO) may play a key role in the beneficial effects of VOO on atherosclerosis. In the present study we evaluated the influence of the unsaponifiable fraction of VOO on the production of eicosanoids and nitric oxide (NO) by endothelial cells (HUVECs). Triglyceride-rich lipoprotein (TRLs) were isolated from human serum after the intake of meals enriched in 3 high-oleic acid oils, i.e., high-oleic sunflower (HOSO), VOO, or enriched-virgin olive (EVO) oils, the last-mentioned containing 2.4% of unsaponifiable matter. HOSO induced a greater accumulation of triglycerides (TGs) in the postprandial serum than VOO or EVO, as measured by calculating the area under the curve. The incubation with TRLs increased NO release by endothelial cells compared with untreated control cells, but the effects of the various TRLs did not differ. EVO-derived TRLs reduced the production of prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TxB(2)) (the stable metabolite of TxA(2)) compared with VOO- or HOSO-derived TRLs. The release of PGI(2) (as 6-keto PGF(1alpha)) was similarly diminished by all TRLs compared with the control. In conclusion, the unsaponifiable fraction of VOO does not affect postprandial triglyceridemia, but it has favorable effects on endothelial function, mainly by reducing proinflammatory and vasoconstrictor eicosanoid synthesis (PGE(2) and TxB(2)).

Evaluation of Improved Coronary Flow Velocity Reserve Using Transthoracic Doppler Echocardiography After Single LDL Apheresis

The purpose of this study was to clarify whether coronary flow velocity reserve (CFVR), evaluated by adenosine 5'-triphosphate-induced hyperemia, is improved by single low-density lipoprotein (LDL) apheresis. Lipid lowering therapy is known to improve endothelium-dependent vasodilatation in forearm or coronary resistant vessels. However, few reports have studied the effect of acute LDL reduction on CFVR. Seven patients with familial hypercholesterolemia and significant coronary stenosis except in the left anterior descending artery (LAD) were enrolled in this study. Coronary flow velocity reserve was estimated before and after LDL apheresis using transthoracic Doppler echocardiography (TTDE), which detects the flow velocity at the distal site of the LAD. Although the averaged diastolic peak velocity (ADPV) during ATP-induced hyperemia was similar before and after LDL apheresis, the ADPV at baseline decreased from 30.69 to 25.56 cm/s, resulting in an increased CFVR from 1.78 to 2.10 (P < 0.001). Plasma bradykinin and 6 keto PGF1alpha increased while fibrinogen and plasma viscosity decreased after apheresis. Single LDL apheresis improves CFVR according to TTDE analysis because of the decreasing ADPV at baseline, which is thought to be induced by epicardial coronary artery dilatation and improved microvessel function. This is the result of various factors, such as changes in plasma LDL cholesterol, bradykinin and PGI2 levels with LDL apheresis.

A polymorphism in the cyclooxygenase 2 gene as an inherited protective factor against myocardial infarction and stroke.

Myocardial infarction (MI) and ischemic stroke are thought to be caused by matrix digestion by metalloproteinases (MMPs) leading to rupture of atherosclerotic plaques. Production of macrophage MMP-2 and MMP-9 is induced by cyclooxygenase 2 (COX-2) and prostaglandin E(2) synthesis. Although COX-2 expression may be genetically determined, the relation between COX-2 polymorphisms and the risk of MI and stroke is unclear. To investigate the relationship between the -765G-->C polymorphism of the COX-2 gene and clinically evident plaque rupture. Prospective, matched case-control study conducted between March 2002 and October 2003 among 864 patients with first MI or atherothrombotic ischemic stroke and 864 hospitalized controls. The groups were matched for age, sex, body mass index, smoking, hypertension, hypercholesterolemia, and diabetes. The -765G-->C variant of the COX-2 gene was genotyped by restriction endonuclease digestion of polymerase chain reaction products. Presence of the -765G-->C polymorphism of the COX-2 gene; COX-2, MMP-2, and MMP-9 expression and activity in plaques and in peripheral monocytes; urinary 6-keto PGF1alpha (marker of endothelial prostacyclin); and endothelium-dependent and -independent forearm blood flow vasodilation. The prevalence of -765GC was 2.41 times higher among controls than among cases (43.3% vs 17.9%; P<.001). The prevalence of -765CC homozygosity was 5.81 times higher (6.4% vs 1.1%; P =.04). Among participants carrying the -765GC and -765CC genotypes, the prevalence ratios for MI or stroke were 0.48 (95% CI, 0.36-0.68) and 0.33 (95% CI, 0.24-0.55), respectively. Expression of COX-2 and MMPs was significantly lower in atherosclerotic plaques from participants carrying the -765C allele, while the -765G-->C polymorphism did not affect endothelial prostacyclin biosynthesis or endothelium-dependent vasodilation in vivo. In subgroup analyses (n = 224 cases), serum high-sensitivity C-reactive protein was significantly lower in patients carrying the -765C allele (mean [SD], 0.78 [0.1] vs 2.56 [0.4] mg/L; P =.04). We found that the -765G-->C polymorphism of the COX-2 gene is associated with a decreased risk of MI and stroke. Detection of this genotype may be useful for predicting genetic risk of MI and stroke.

Effects of nitric oxide and prostacyclin on hemodynamic response by big endothelin-1 in near term fetal sheep

To measure plasma concentrations of endothelin (ET)-1, NO metabolites (nitrate/nitrite; NOx) and 6-keto PGF1 alpha (PGF1 alpha) in maternal and fetal sheep blood, and to evaluate the effects of big ET-1 on hemodynamic response, blood gases and NO and 6-keto PGF1 alpha production in near term fetal sheep. Hemodynamic parameters were measured during infusion of big ET-1 into the carotid vein in chronically catheterized fetal sheep on day 125 of gestation. Fetal arterial blood samples were obtained for ET-1, PGF1 alpha) and nitrate/nitrite (NOx) measurements. ET-1, NOx and PGF1 alpha plasma concentrations were all significantly higher in fetal compared with the maternal plasma. Big ET-1 significantly decreased fetal systolic and diastolic blood pressure and significantly increased fetal heart rate. Big ET-1 stimulated plasma PGF1 alpha), but not NOx , concentration. Circulatory regulating factors in the fetus were up-regulated. The effects of ET-1 on fetal hemodynamic response may be mediated via prostacyclin, but not via the NO pathway.

Inhibition of COX-2 prevents hypertension and proteinuria associated with a decrease of 8-iso-PGF2alpha formation in L-NAME-treated rats.

The inhibition of nitric oxide (NO) exerts injurious effects on the cardiovascular system by several mechanisms, such as the activation of the renin-angiotensin system, oxidative stress, and inflammatory cytokines. We examined whether COX-2, an inducible isoform of cyclooxygenase, is associated with the pathogenesis observed in N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Three groups of 8-week-old male Sprague-Dawley rats were studied (n = 6 in each group): group 1, untreated controls; group 2, treated with L-NAME (1 g/l for 3 weeks, p.o.); and group 3, L-NAME co-treated with COX-2 inhibitor NS-398 (5 mg/kg per day, i.p.). The L-NAME-induced expression of COX-2 mRNA and protein was semi-quantified in the kidneys and the thoracic aorta. Urinary excretion of the prostaglandin 6-keto PGF(1alpha), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2) was measured in the three groups. Moreover, urinary excretion of 8-iso-PGF(2alpha), a potent vasoconstricting arachidonic acid metabolite acting through thromboxane A (TXA) receptor activation, proposed recently as a marker of oxidative stress, was also measured. L-NAME induced significant increases in systolic blood pressure (P< 0.01), urinary protein (P< 0.05), and renal excretion of 8-iso-PGF(2alpha)(P< 0.01), compared with the control. In L-NAME-treated rats, the levels of COX-2 mRNA and protein were more than 50% higher in the kidneys (P< 0.05), and six-fold higher in the thoracic aorta (P< 0.01) than in control rats. NS-398 significantly ameliorated an increase in systolic blood pressure (P< 0.01) and urinary protein (P< 0.05) induced by L-NAME. These data indicate that an increase in COX-2 expression might have a hypertensive effect, partly associated with 8-iso-PGF(2alpha) formation in l-NAME-treated rats.

Prostaglandins contribute to impaired angiotensin II-induced cerebral vasodilation after brain injury.

This study characterized the effects of fluid percussion brain injury (FPI) on angiotensin II (AII)-induced cerebral vasodilation, determined the role of prostaglandins in such changes and evaluated the contribution of two subtypes of AII receptors (AT(1) and AT(2)) to the effects of AII on cerebrovascular regulation. Topical AII (10(-8), 10(-6), 10(-4) M) elicited vasodilation, which was attenuated by FPI (10 +/- 1; 18 +/- 2; 27 +/- 1% vs. 2 +/- 1; 4 +/- 1; 7 +/- 1%). Such changes in diameter were associated with increases in CSF 6-keto-PGF(1alpha), the stable breakdown product of PGI(2) (1.5 +/- 0.1; 2.1 +/- 0.1; 4.0 +/- 0.3 fold) and TXB(2), the stable breakdown product of TXA(2) (1.2 +/- 0.1; 1.4 +/- 0.1; 1.6 +/- 0.1 fold). However, after FPI, increases in 6-keto PGF(1alpha) were blocked (1.0 +/- 0.1; 1.0 +/- 0.1; 1.1 +/- 0.1 fold) whereas TXB(2) release was enhanced (1.5 +/- 0.1; 1.8 +/- 0.1; 1.9 +/- 0.1 fold). Pretreatment with the cyclooxygenase inhibitor indomethacin (5 mg/kg i.v.) in FPI animals partially protected AII vasodilation (8 +/- 1; 14 +/- 2; 19 +/- 3%). CGP 42112A, a putative AT(2) agonist, elicited vasodilation, which was also blunted by FPI. Such dilation was not associated with CSF prostaglandin changes, and indomethacin did not protect responses altered by FPI. Vasodilatation caused by low concentrations of AII was blunted by an AT(1) antagonist ZD 7155 but unchanged by an AT(2) antagonist PD 123,319. The high AII concentration produced dilation that was blunted by both antagonists. These data show that FPI impairs AII-mediated vasodilation. These data suggest that FPI causes these changes via alteration in an AT(1)-mediated production of prostaglandins. These data additionally suggest that FPI induced impairment of AT(2) mediated vasodilation is independent of an altered production of prostaglandins.

Interactions of adenosine, prostaglandins and nitric oxide in hypoxia-induced vasodilatation: in vivo and in vitro studies.

Adenosine, prostaglandins (PG) and nitric oxide (NO) have all been implicated in hypoxia-evoked vasodilatation. We investigated whether their actions are interdependent. In anaesthetised rats, the PG synthesis inhibitors diclofenac or indomethacin reduced muscle vasodilatation evoked by systemic hypoxia or adenosine, but not that evoked by iloprost, a stable analogue of prostacyclin (PGI(2)), or by an NO donor. After diclofenac, the A(1) receptor agonist CCPA evoked no vasodilatation: we previously showed that A(1), but not A(2A), receptors mediate the hypoxia-induced muscle vasodilatation. Further, in freshly excised rat aorta, adenosine evoked a release of NO, detected with an NO-sensitive electrode, that was abolished by NO synthesis inhibition, or endothelium removal, and reduced by ~50 % by the A(1) antagonist DPCPX, the remainder being attenuated by the A(2A) antagonist ZM241385. Diclofenac reduced adenosine-evoked NO release by ~50 % under control conditions, abolished that evoked in the presence of ZM241385, but did not affect that evoked in the presence of DPCPX. Adenosine-evoked NO release was also abolished by the adenyl cyclase inhibitor 2',5'-dideoxyadenosine, while dose-dependent NO release was evoked by iloprost. Finally, stimulation of A(1), but not A(2A), receptors caused a release of PGI(2) from rat aorta, assessed by radioimmunoassay of its stable metabolite, 6-keto PGF(1alpha), that was abolished by diclofenac. These results suggest that during systemic hypoxia, adenosine acts on endothelial A(1) receptors to increase PG synthesis, thereby generating cAMP, which increases the synthesis and release of NO and causes muscle vasodilatation. This pathway may be important in other situations involving these autocoids.

Cyclo-oxygenase-2 inhibition increases blood pressure in rats.

1 It is known that nonselective cyclo-oxygenase (COX) inhibitors have small but significant effects on blood pressure (BP), most notably in hypertensive patients on antihypertensive medication. Whether selective COX-2 inhibitors also interfere with BP regulation is not well understood. Therefore, we aimed to examine the effect of chronic treatment with a selective COX-2 inhibitor (rofecoxib) on systolic blood pressure (sBP) in normotensive Wistar-Kyoto rats (WKY) and on the developmental changes of sBP in young spontaneously hypertensive rats (SHR). In addition, we investigated a possible influence of salt intake on the effects of COX-2 inhibition on BP in these two rat strains. 2 Rofecoxib dose dependently increased sBP and decreased plasma levels of 6-keto prostaglandin (PG)F(1alpha) in WKY rats fed a normal salt diet (0.6% NaCl, wt wt(-1)), without affecting serum thromboxane (TX)B(2) levels. 3 Rofecoxib significantly elevated sBP in both rat strains fed normal salt or high salt diet (8% NaCl, wt wt(-1)), but not in rats on low salt intake (0.02% NaCl, wt wt(-1)). 4 Rofecoxib significantly decreased plasma levels of 6-keto PGF(1alpha) in both rat strains fed normal or high salt diet, but not in rats during low salt intake. 5 Rofecoxib exerted no influence on the changes of body weight nor on water intake. Plasma renin activity (PRA) and renocortical renin mRNA abundance were not changed by rofecoxib, but plasma aldosterone concentration (PAC) was significantly reduced. 6 These results suggest that chronic inhibition of COX-2 causes an increase of blood pressure that depends on prostacyclin synthesis. Furthermore, this increase is independent on genetic predisposition and can be prevented by salt deprivation. Since water intake and body weight gain were not changed by rofecoxib, fluid retention appears not to be a major reason for the development of hypertension. Similarly, an activation of the renin-angiotensin-aldosterone axis appears to be an unlikely candidate mechanism.

Angiotensin II Increases Leptin Secretion by 3T3-L1 and Human Adipocytes via a Prostaglandin-Independent Mechanism

We previously reported that angiotensin II (Ang II) increases adipocyte fatty acid synthesis and triglyceride content. Triglyceride stores or adiposity correlate positively with the amount of circulating leptin. Ang II was proposed to increase adipocyte differentiation and growth by promoting prostaglandin (PG) production. The purpose of this study was to determine whether Ang II increases leptin secretion via a PG-dependent mechanism. Physiologic doses of Ang II significantly increased leptin secretion by 3T3-L1 adipocytes and human adipocytes. Elevation of PG secretions was elicited at physiologic concentrations of Ang II (P < 0.05). Secretions of 6-keto PGF(1alpha), a stable derivative of PGI(2), and PGE(2) were induced by physiologic concentrations of Ang II in a time-responsive fashion (P < 0.05). Inhibition of PG synthesis by indomethacin and aspirin significantly suppressed basal as well as Ang II-induced PG levels, but did not significantly affect basal and Ang II-induced leptin secretion. In conclusion, although Ang II stimulates both leptin and PG secretion by adipocytes, regulation of leptin secretion by Ang II in adipocytes is not mediated by a PG-dependent mechanism.

17-beta-oestradiol-induced vasorelaxation in vitro is mediated by eNOS through hsp90 and akt/pkb dependent mechanism.

1. The L-arginine-NO pathway has been implicated in the vasorelaxant effect of 17-beta-oestradiol. Here we have addressed the involvement of two distinct activation steps of endothelial nitric oxide synthase (eNOS) in the 17-beta-oestradiol-induced vasorelaxant effect on rat aortic rings. 2. Rat aortic rings contracted with phenylephrine (PE) 1 microM relaxed in a concentration related fashion to 17-beta-oestradiol water soluble cyclodextrin-encapsulated (E2) only when endothelium was present. The pure anti-oestrogen of E2 receptor ICI 182,780 (20 microM) significantly inhibited E2-induced vasorelaxation. 3. Geldanamycin (10 microM), a specific inhibitor of heat shock protein 90 (hsp90) and N(omega)-nitro-L-arginine-methyl ester (L-NAME, 100 microM), a nitric oxide synthase inhibitor, significantly inhibited E2-induced vasorelaxation. 4. Incubation of rat aortic rings up to 6 h with LY 294002 (25 microM), a specific inhibitor of PI(3)K akt/pkb pathway reduced E2-induced vasorelaxation. 5. Incubation of rat isolated aorta with E2, induced prostacyclin (PGI(2)) release. PGI(2) levels, measured as 6-keto PGF(1alpha), were abolished by ibuprofen (10 microM), both L-NAME and GA did not influence basal or E2-stimulated PGI(2) confirming the specificity of these two compounds on eNOS pathway. 6. In conclusion, we demonstrate that E2 interaction with its receptor is followed by a vasorelaxant effect in rat aortic rings mediated by eNOS activation through both hsp90 and akt/pkb dependent mechanisms.

Serotonin 5-HT1 Receptors Potentiate Histamine and Thrombin Stimulated Prostaglandin Synthesis in Endothelial Cells

The ability of serotonin 5-HT1 receptors to increase vascular tone was previously found to be activated by vasoconstrictiors such as histamine. In this study, treatment of cultured human aortic endothelial cells (HAEC) with the 5-HT1-selective agonist 5-carboxamidotryptamine (5-CT) alone had no effect on the levels of prostaglandin F2alpha (PGF2alpha) or 6-keto-prostaglandin F1alpha (6-keto PGF1alpha). However, 5-CT potentiated the histamine and thrombin stimulated increases in prostaglandins released by HAEC. In the presence of histamine, increasing doses of 5-CT caused a steep rise in PGF2alpha levels resulting in an increase in the ratio of PGF2alpha over 6-keto PGF1alpha. The ability of 5-CT to potentiate prostaglandin production was correlated with its ability to potentiate the histamine and thrombin mediated mobilization of arachidonic acid. These results demonstrate that the ability of 5-HT1 receptors to stimulate prostaglandin production in endothelial cells is activated by histamine and thrombin.

Nitric oxide metabolites, prostaglandins and trigeminal vasoactive peptides in internal jugular vein blood during spontaneous migraine attacks.

Despite evidence emerging from the experimental model of nitroglycerin-induced headache, the endogenous increase in nitric oxide (NO) production during migraine attacks is only speculative. It has been hypothesized that there is a close relationship between activation of the L-arginine/NO pathway and production of certain vasoactive and algogenic prostaglandins during spontaneous migraine attacks, but this suggestion also needs to be confirmed. In the present study the levels of nitrites, the stable metabolites of NO, were determined with high performance liquid chromatography (HPLC) in the internal jugular venous blood of five patients affected by migraine without aura examined ictally. These samples were taken within 30 min, 1, 2, and 4 h from the onset of the attack and at the end of the ictal period. At the same time, the plasma levels of calcitonin gene-related peptide (CGRP), neurokinin A (NKA), prostaglandin E2 (PGE2) and 6 keto PGF1alpha, the stable product of PGI2, were assessed with radioimmunoassay (RIA) kits in the same samples. The levels of the intracellular messengers, cGMP and cAMP, were also measured with the RIA method. Nitrite, cGMP, CGRP and NKA levels reached their highest values at the first hour, then they tended to decrease progressively and returned, after the end of attacks, to values similar or below those detected at the time of catheter insertion (ANOVA, statistical significance: P<0.001; P<<0.002; P<0.002; P<0.003, respectively). PGE2 and 6 keto PGF1alpha, as well as cAMP levels also significantly increased at the first hour but reached a peak at the 2nd hour and remained in the same range until the 4th and 6th hours. Then their values tended to decrease after the end of attacks, becoming lower than those measured immediately after catheter positioning for internal jugular venous blood drawing (ANOVA: P<0.002, P<0.004, P<0.001, respectively). Our results support early activation of the L-arginine/NO pathway which accompanies the release of vasoactive peptides from trigeminal endings and a late rise in the synthesis of prostanoids with algogenic and vasoactive properties which may intervene in maintaining the headache phase.

The effect of intensive plasma water exchange by hemofiltration on hemodynamics and soluble mediators in canine endotoxemia.

High volume hemofiltration (HVHF) (200 ml/kg/h) improves hemodynamics in experimental septic shock but is difficult to apply clinically. Accordingly, we studied whether less intensive HVHF (80 ml/kg/h) can still improve hemodynamics in experimental septic shock. We also investigated its effect on the serum concentrations of several inflammatory mediators, including endothelin (ET-1), endotoxin (LPS), tumor necrosis factor-alpha (TNF-alpha), and 6-keto prostaglandin F(1alpha) (6-kepto PGF(1alpha)). Sixteen anesthetized dogs were connected to a continuous veno-venous hemofiltration (CVVH) (filtration: 80 ml/kg/h) or sham circuit and endotoxin (0.5 mg/kg) was infused intravenously over 5 min. Hemodynamic variables were measured at baseline and at 15, 45, 90, and 180 min. The major hemodynamic finding was that endotoxin-induced hypotension was significantly attenuated by intensive CVVH (p < 0.04). Changes in cardiac output and right ventricular ejection fraction were equal in both groups. ET-1 levels, but not LPS, TNF-alpha, or 6-keto PGF(1alpha), were lower during CVVH (p = 0.042). Endotoxin or TNF-alpha were not found in the ultrafiltrate. Median clearances of ET-1 and 6-keto PGF(1alpha) during intensive CVVH were 8.8 and 25.9 ml/m, respectively. We conclude that intensive CVVH attenuates the early component of endotoxin-induced hypotension and reduces serum concentrations of endothelin-1. The effect of CVVH on blood pressure is not explained by convective clearance of the mediators in question.

Selective cyclo‐oxygenase‐2 inhibition with celecoxib elevates blood pressure and promotes leukocyte adherence

1. Selective inhibitors of cyclo-oxygenase-2 have been shown to be effective anti-inflammatory drugs with reduced gastrointestinal toxicity relative to conventional nonsteroidal anti-inflammatory drugs (NSAIDs). In the present study, we examined the possibility that selective COX-2 inhibition, by blocking prostacyclin synthesis, would increase blood pressure and cause leukocyte adherence and platelet aggregation. 2. Normal rats and rats with hypertension induced by chronic administration of Nomega-nitro-L-arginine methylester were given celecoxib (10 mg kg(-1)) daily for 3 weeks. Celecoxib significantly elevated of blood pressure in both the normal and hypertensive rats (mean increase of >33 mm Hg after 3 weeks). 3. In normal rats, celecoxib had no effect on serum 6-keto prostaglandin (PG)F(1alpha) levels. Hypertensive rats exhibited a significant increase (82%) in serum 6-keto PGF(1alpha) levels, and this was reduced to the levels of normal rats by treatment with celecoxib. 4. Rats treated with celecoxib exhibited significant increases in weight gain (20%), plasma arginine-vasopressin levels (148%) and plasma urea (69%) relative to vehicle-treated controls. Plasma creatinine levels were unaffected by treatment with celecoxib, while plasma renin levels were significantly decreased (30%) relative to controls. 5. Superfusion of mesenteric venules with celecoxib (3 microM) in vivo resulted in significant increases in leukocyte adherence to the endothelium in both normal and hypertensive rats. 6. These studies suggest that suppression of COX-2 significantly influences vascular and/or renal function, leading to elevated blood pressure and leukocyte adherence.

Dietary n-3 fatty acids, curcumin and capsaicin lower the release of lysosomal enzymes and eicosanoids in rat peritoneal macrophages.

Male Wistar rats (12 rats/group) were fed a diet containing 8 wt % coconut oil or groundnut oil or cod-liver oil for a total period of 8 weeks. The diets were also supplemented with 2 wt % groundnut oil for providing essential fatty acids. During the last 2 weeks, 6 rats form each group were additionally given curcumin (30 mg/kg body wt/day) or capsaicin (5 mg/kg body wt/day) in 1 ml groundnut oil. The peritoneal macrophages from rats fed cod-liver oil diet secreted lower levels of lysosomal enzymes collagenase, elastase and hyaluronidase as compared to those from rats fed coconut oil or groundnut oil diets. Curcumin and capsaicin significantly lowered the secretion of these lysosomal enzymes from macrophages in animals given coconut oil or groundnut oil diet. Macrophages from rats fed cod-liver oil secreted lower amounts of prostaglandin E2, 6-keto PGF1alpha, leukotrienes B4 and C4 and also incorporated lesser amounts of [3H]-arachidonic acid as compared to those given coconut oil or groundnut oil diets. Curcumin and capsaicin lowered the secretion of these eicosanoids and decreased the incorporation of [3H]-arachidonic acid in macrophage lipids. However curcumin and capsaicin significantly increased the secretion of 6-keto PGF1alpha in all the groups of animals. These studies indicated that dietary cod-liver oil (rich in n-3 fatty acids), and spice principles curcumin and capsaicin can lower the secretory functions of macrophages in a beneficial manner.

Interleukin‐8 Stimulates Placental Prostacyclin Production in Preeclampsia

Our purpose was to determine placental interleukin (IL)-8 production and its correlation with the prostacyclin production in normal and preeclamptic pregnancies and to evaluate the beneficial effect of IL-8 on prostacyclin production. We determined 1) the in vitro production of IL-8 and prostacyclin by placental villous tissues from normal and preeclamptic pregnancies and 2) the production of prostacyclin by villous tissues from preeclampsia treated with recombinant human IL-8 (rhIL-8). IL-8 levels were measured by enzyme-linked immunosorbent assay and prostacyclin by radioimmunoassay of 6-keto PGF1alpha, the stable metabolite of prostacyclin. 1) Placental production of IL-8 and 6-keto PGF1alpha were significantly less in preeclampsia than in normal pregnancies, P<0.05. 2) Placental production of 6-keto PGF1alpha and IL-8 was significantly correlated in preeclampsia, P<0.01. 3) Placental tissues treated with IL-8 exhibited a concentration-dependent increase in 6-keto PGF1alpha production. Placental tissues from preeclampsia produce significantly less IL-8 than tissues from normal pregnancies, which correlates with decreased prostacyclin production. IL-8 improves placental prostacyclin production in preeclampsia.

Estrogen decreases prostaglandin H synthase products from endothelial cells.

Because we showed recently that estrogen replacement prevents prostaglandin H synthase (PGHS)-dependent vasoconstriction in rats, the aim of this study was to determine how estradiol affects production of PGHS-dependent eicosanoids. Cultured bovine coronary microvascular endothelial cells were exposed to physiologic levels of 17 beta-estradiol (0.01 nM [about 2.7 pg/mL], 0.1 nM [about 27 pg/mL], or 1.0 nM [about 270 pg/mL]) for 4, 8, or 24 hours. Thromboxane (TXA2), prostacyclin (PGI2), and nitric oxide (NO) were measured as their stable metabolites, thromboxane B2 (TXB2), 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), and nitrite (NO2), respectively. Estradiol had no effect on nitrite production. However, exposure to 0.1 nM and 1.0 nM estradiol for 24 hours reduced TXB2 production to 67 +/- 16% and 69 +/- 12% of control, respectively. Furthermore, 0.1 nM and 1.0 nM estradiol also reduced production of 6-keto PGF1 alpha to 35 +/- 19% and 17 +/- 11% of control, respectively. Prostaglandin H synthase expression was not altered by estradiol. However, the estrogen receptor inhibitor, tamoxifen, reversed the inhibitory effect of estradiol. Estradiol acts through a receptor-dependent process to decrease PGHS-dependent products, thus further elucidating this novel effect of estradiol on the vascular system.

Eicosanoid release in the endotoxin-primed isolated perfused rat lung and its pharmacological modification

Recent observations have demonstrated a central role of the "inducible" isoform of the cyclooxygenase (COX), COX-2, in the rat lung. Therefore, the reported capacity of selective COX-2 inhibitors to potentiate the formation of leukotriene (LT) B4 may raise concern about pro-inflammatory side effects of such drugs in the respiratory system. The present study was aimed at determining the effects of the COX-2 inhibitor NS-398 on the release of COX and 5-lipoxygenase (LOX) metabolites of arachidonic acid in isolated perfused lungs obtained from endotoxin-treated rats before and after stimulation with the leukocyte secretagogue N-formyl-methionyl-leucyl-phenylalanine (FMLP). Two hours after rats had received endotoxin i.v., the lung was dissected and perfused via the pulmonary artery with physiological salt solution. After an equilibration period of 20 min the outflow was collected (5-min fractions). In the respective treatment groups, indomethacin, NS-398, or the 5-LOX inhibitor MK886 were present throughout the experiment, while FMLP was added to the perfusate during a single 5-min period. The concentration of eicosanoids in the outflow was determined by radioimmunoassay. Endotoxin treatment of rats resulted in increased expression of COX-2 mRNA in lung tissue, and an elevated basal release of the prostaglandin (PG)I2 metabolite 6-keto PGF1alpha, without a detectable increase of leukotriene (LT) formation. In-vitro exposure to FMLP stimulated LT and prostanoid release, which was significantly enhanced in endotoxin-primed lungs, and was suppressed by the 5-LOX inhibitor MK-886 (3 microM) and the COX-inhibitor indomethacin (5 microM), respectively. Either compound showed selective inhibition of the respective pathway of arachidonic acid metabolism. In endotoxin-primed lungs, the COX-2 inhibitor NS-398 (0.3-1.0 microM) depressed basal as well as FMLP-stimulated release of 6-keto PGF1alpha, but did not cause a significant increase of LTB4 or cysteinyl-LT release. These results suggest that FMLP, presumably acting on inflammatory cells trapped in the pulmonary circulation of endotoxin treated rats, induced prostanoid formation mainly via the COX-2 pathway, and that its inhibition by NS-398 had no detectable potentiating effect on LTB4 or cysteinyl-LT biosynthesis.