The effect of kisspeptin on the regulation of vascular tone.

Kisspeptin has been implicated in cardiovascular control. Eicosanoids play a crucial role in the activation of platelets and the regulation of vascular tone. In the present study, we investigated the effect of kisspeptins on eicosanoid synthesis in platelets and aorta in vitro. Platelets and aorta were isolated from Wistar-Kyoto rats. After preincubation with different doses of kisspeptin, samples were incubated with [1-(14)C]arachidonic acid (0.172 pmol/mL) in tissue culture Medium 199. The amount of labeled eicosanoids was measured with liquid scintillation, after separation with overpressure thin-layer chromatography. Kisspeptin-13 stimulated the thromboxane synthesis. The dose-response curve was bell-shaped and the most effective concentration was 2.5 × 10(-8) mol/L, inducing a 27% increase. Lipoxygenase products of platelets displayed a dose-dependent elevation up to the dose of 5 × 10(-8) mol/L. In the aorta, kisspeptin-13 induced a marked elevation in the production of 6-keto-prostaglandin F1α, the stable metabolite of prostacyclin, and lipoxygenase products. Different effects of kisspeptin on cyclooxygenase and lipoxygenase products indicate that beyond intracellular Ca(2+) mobilization, other signaling pathways might also contribute to its actions. Our data suggest that kisspeptin, through the alteration of eicosanoid synthesis in platelets and aorta, may play a physiologic and (or) pathologic role in the regulation of vascular tone.

Effects of carboxymethylpachymaran on signal molecules in chicken immunocytes.

The study was carried out to investigate the immunomodulation mechanism of carboxymethylpachymaran (CMP). Chicken splenic lymphocytes were cultured in medium alone or with CMP at the final concentration of 50mg/L, 100mg/L, 200mg/L or 400mg/L in vitro for 4h, 8h, 12h or 24h, respectively. The supernatants at different culture periods were analyzed for changes in levels of 6-keto-prostaglandin F1α (6-keto-PGF1α), thromboxane B2 (TXB2) and nitric oxide (NO). The cells were collected to determine contents of oxidized glutathione (GSSG), reduced glutathione (GSH), cyclic AMP (cAMP) and cyclic GMP (cGMP). The results showed that CMP increase the values of NO, 6-keto-PGF1α, TXB2, and the ratio of 6-keto-PGF1α to TXB2 in supernatants. The contents of intracellular GSH, cAMP, cGMP and the ratio of cAMP to cGMP were increased in the cells treated with CMP. The results suggested that CMP enhanced immune functions by increasing the contents of GSH and by regulating arachidonic acid signal transduction systems in chicken splenic lymphocytes. The signal pathway of NO-cGMP plays an important role in CMP-induced activation of chicken splenic lymphocytes.

Effects of selective and non-selective cyclooxygenase inhibition against neurological deficit and brain oedema following closed head injury in mice.

The implication of cyclooxygenase (COX) type 2 in post-traumatic consequences is so far controversial. In experimental models of traumatic brain injury (TBI), genetic disruption or pharmacological inhibition of COX-2 has been shown to be neuroprotective, deleterious or without effect. Therefore, the aim of our study was to investigate the effect of COX-2 inhibition against neurological deficit and brain oedema after TBI that was induced by mechanical percussion in male Swiss mice. Despite the increased level and activity of COX-2, its inhibition either with nimesulide (12 mg/kg) or meloxicam (2mg/kg) modified neither the neurological score nor the brain water content that were evaluated at 6 and 24h after injury. Interestingly, the non-selective COX inhibition with indomethacin (5mg/kg) significantly promoted neurological recovery at 6 and 24h after trauma, without improving brain oedema. In conclusion, the present study yields considerable evidence that COX-2 may not solely constitute an interesting target for the treatment of TBI consequences. Our data point to a potentially deleterious role of COX-1 in the development of neurological impairment in brain-injured mice. However, the neuroprotective mechanism of indomethacin remains to be clarified.

Niacin and biosynthesis of PGD2by platelet COX-1 in mice and humans.

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD2 and PGE2 followed by COX-2-dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2-derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

Resistance artery mechanics and composition in angiotensin II-infused mice: effects of cyclooxygenase-1 inhibition.

AIMS: The aim of this study was to investigate the role of cyclooxygenase (COX)-1 on vascular alterations in structure, mechanics, and extracellular matrix (ECM) components induced by angiotensin (Ang) II in mesenteric arteries from wild-type (WT) and COX-1 knockout (COX-1(-/-)) mice. METHODS AND RESULTS: Animals were infused with vehicle or Ang II (400 ng/kg/min, s.c.) ± SC-560 (COX-1 inhibitor), DFU (COX-2 inhibitor), or SQ-29548 (TP receptor antagonist). After 2 weeks, vessels were isolated and exposed to intraluminal pressures (3-140 mmHg, pressurized myograph) to determine mechanical properties. Angiotensin II-induced vascular hypertrophic remodelling in WT was reversed by SC-560 or SQ-29548, but unaffected by DFU. Angiotensin II increased vessel stiffness (P< 0.01), this effect being ameliorated by SC-560 or SQ-29548, but unmodified by DFU. Angiotensin II failed to modify vessel elasticity in COX-1(-/-) mice. In WT vessels, Ang II enhanced COX-1 immunostaining, induced collagen and fibronectin depositions and decreased elastin content (P< 0.01). These effects were reversed by SC-560 or SQ-29548, but unaffected by DFU. In COX-1(-/-) mice, Ang II did not affect ECM contents. In WT, Ang II increased COX-1 and decreased COX-2 expression, and enhanced the vascular release of 6-keto-PGF1α which was prevented by COX-1 blockade. Human coronary artery smooth muscle cells, incubated with Ang II, showed an increased expression of procollagen I, which was abrogated by SC-560 or SQ-29548. CONCLUSION: Angiotensin II-induced alterations of resistance arteries in structure, mechanics, and ECM composition were prevented by COX-1 inhibition and TP receptor antagonism, indicating that Ang II-mediated vascular damage is mediated by COX-1-derived prostanoid prostacyclin, activating TP receptors.

Chemical composition of hepatic lipids mediates reperfusion injury of the macrosteatotic mouse liver through thromboxane A(2).

BACKGROUND & AIMS: Chemical composition of hepatic lipids is an evolving player in steatotic liver ischemia/reperfusion (I/R) injury. Thromboxane A(2) (TXA(2)) is a vasoactive pro-inflammatory lipid mediator derived from arachidonic acid (AA), an omega-6 fatty acid (Ω-6 FA). Reduced tolerance of the macrosteatotic liver to I/R may be related to increased TXA(2) synthesis due to the predominance of Ω-6 FAs. METHODS: TXA(2) levels elicited by I/R in ob/ob and wild type mice were assessed by ELISA. Ob/ob mice were fed Ω-3 FAs enriched diet to reduce hepatic synthesis of AA and TXA(2) or treated with selective TXA(2) receptor blocker before I/R. RESULTS: I/R triggered significantly higher hepatic TXA(2) production in ob/ob than wild type animals. Compared with ob/ob mice on regular diet, Ω-3 FAs supplementation markedly reduced hepatic AA levels before ischemia and consistently blunted hepatic TXA(2) synthesis after reperfusion. Sinusoidal perfusion and hepatocellular damage were significantly ameliorated despite downregulation of heme oxygenase-1. Hepatic transcript and protein levels of IL-1ß and neutrophil recruitment were significantly diminished after reperfusion. Moreover, TXA(2) receptor blockage conferred similar protection without modification of the histological pattern of steatosis. A stronger protection was achieved in the steatotic compared with lean animals. CONCLUSIONS: Enhanced I/R injury in the macrosteatotic liver is explained, at least partially, by TXA(2) mediated microcirculatory failure rather than size-related mechanical compression of the sinusoids by lipid droplets. TXA(2) blockage may be a simple strategy to include steatotic organs and overcome the shortage of donor organs for liver transplantation.

Sodium tungstate and vanadyl sulfate effects on blood pressure and vascular prostanoids production in fructose-overloaded rats.

This study analyzes the effects of sodium tungstate and vanadyl sulphate in the fructose-overloaded rat, a model of metabolic syndrome. Fructose (9 weeks) increased blood pressure, triglycerydemia, glycemia, and reduced release of vasodilator prostaglandins (prostacyclin and prostaglandin E2 ) in the mesenteric vascular bed. Sodium tungstate prevented those alterations; meanwhile vanadyl sulfate only prevented the increase in glycemia. In conclusion, the present experiments showed that sodium tungstate is more effective than vanadyl sulfate for the treatment of experimental metabolic syndrome in rats.

Initial response of endothelial cells to acute stimulation with a lipid component: increase cyclooxygenase activity by induction of COX-2 through activation of tyrosine kinase.

OBJECTIVE: To study the initial response of endothelial cells acutely stimulated with a lipid component in the aspect of cyclooxygenase (COX) function which needed for prostacyclin synthesis, an endogenous antiatherogenic agent secreted from endothelial cells. MATERIAL AND METHOD: 25 hydroxycholesterol (25OHC) was used as a representative lipid component for stimulating human umbilical vein endothelial cell (HUVEC) obtained from umbilical cords of healthy newborns with informed consent of their mothers. HUVEC were treated with 25OHC (0.1, 1 or 10 microgram/mL) at times 6, or 24 h. COX activity was measured from amount of 6-keto-PGF(1 alfa) production in the presence of exogenous arachidonic acids (10 micromolar; 10 min) by enzyme immunoassay. The amount of COX-1 and COX-2 protein were detected by Western blot. Cell viability was assessed by using MTT assay. RESULTS: 25OHC induced COX-2 protein production with increasing the activity of COX enzyme in HUVEC without change in amount of COX-1 protein. The induction of COX-2 or increasing in COX activity depended on concentration of 25OHC and time to exposure which seemed to be inhibited by genistein, a specific tyrosine kinase inhibitor. CONCLUSION: Acute stimulation of HUVEC with 25OHC, an atherosclerotic lipid component, increases the activity of COX by inducing COX-2 expression in a manner that depended on concentration and time. The induction of COX-2 expression might possibly mediated through activation protein tyrosine kinase. These responses may be an initial defensive mechanism of endothelial cells from lipid component attack.

Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1.

Prostaglandin E(2) (PGE(2)) plays a crucial role in the apparent link between tumor growth and chronic inflammation. Cyclooxygenase (COX)-2 and microsomal PGE(2) synthase-1, which are overexpressed in many cancers, are functionally coupled and thus produce massive PGE(2) in various tumors. Curcumin, a polyphenolic beta-diketone from tumeric with anti-carcinogenic and anti-inflammatory activities, was shown to suppress PGE(2) formation and to block the expression of COX-2 and of microsomal PGE(2) synthase-1. Here, we identified microsomal PGE(2) synthase-1 as a molecular target of curcumin and we show that inhibition of microsomal PGE(2) synthase-1 activity is the predominant mechanism of curcumin to suppress PGE(2) biosynthesis. Curcumin reversibly inhibited the conversion of PGH(2) to PGE(2) by microsomal PGE(2) synthase-1 in microsomes of interleukin-1beta-stimulated A549 lung carcinoma cells with an IC(50) of 0.2 to 0.3 micromol/L. Closely related polyphenols (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic acid) failed in this respect, and isolated ovine COX-1 and human recombinant COX-2 were not inhibited by curcumin up to 30 micromol/L. In lipopolysaccharide-stimulated human whole blood, curcumin inhibited COX-2-derived PGE(2) formation from endogenous or from exogenous arachidonic acid, whereas the concomitant formation of COX-2-mediated 6-keto PGF(1)alpha and COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was suppressed only at significant higher concentrations. Based on the key function of PGE(2) in inflammation and carcinogenesis, inhibition of microsomal PGE(2) synthase-1 by curcumin provides a molecular basis for its anticarcinogenic and anti-inflammatory activities.

PPAR activators and COX inhibitors selectively block cytokine-induced COX-2 expression and activity in human aortic smooth muscle cells.

Atherosclerotic complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells (SMCs), and an accumulation of inflammatory cells. Regulation of this inflammatory response is an essential element in chronic inflammatory diseases such as atherosclerosis. Nuclear receptors and particularly peroxisome proliferator-activated receptors (PPARs) have emerged as therapeutic targets with a widespread impact on the treatment of metabolic disorders because they can modulate gene expression involved in lipid and glucose homeostasis and can exert anti-inflammatory properties. However, little is known about nuclear receptor effects on SMC inflammation, which produces large amounts of IL-6 and prostanoids. The aim of this study was to evaluate anti-inflammatory properties of nuclear receptor activators in a human physiological SMC model. We show that PPAR activators, as well as liver X receptor alpha, farnesoid X receptor and retinoid X receptor alpha activators, inhibit IL-1beta-induced SMC 6-keto PGF1alpha synthesis, an index of cyclooxygenase (COX)-2 activity, with IC(50) between 1 and 69 microM. In contrast, PPARgamma activators, as exemplified by rosiglitazone and pioglitazone, were unable to inhibit cytokine-induced 6-keto PGF1alpha synthesis. We also demonstrate for the first time that the COX-2 inhibitor rofecoxib can reduce 6-keto PGF1alpha production by both enzymatic inhibition and transcriptional repression. These results show that some nuclear receptor activators have SMC anti-inflammatory properties due to COX-2 inhibition which could participate in their anti-atherosclerotic properties beyond lipid impacts.

Myrtucommulone, a natural acylphloroglucinol, inhibits microsomal prostaglandin E(2) synthase-1.

BACKGROUND AND PURPOSE: The selective inhibition of prostaglandin (PG)E(2) formation via interference with microsomal PGE(2) synthase (mPGES)-1 could have advantages in the treatment of PGE(2)-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1. EXPERIMENTAL APPROACH: The effect of MC on PGE(2) formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1beta-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF(1alpha) formation. KEY RESULTS: MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH(2) to PGE(2) (IC(50) = 1 micromol x L(-1)). PGE(2) formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 micromol x L(-1), and only moderate inhibition of cellular or cell-free COX-1 was evident (IC(50) > 15 micromol x L(-1)). CONCLUSIONS AND IMPLICATIONS: MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE(2) formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.

Virgin olive oil polyphenol hydroxytyrosol acetate inhibits in vitro platelet aggregation in human whole blood: comparison with hydroxytyrosol and acetylsalicylic acid.

Hydroxytyrosol acetate (HT-AC) is a polyphenol present in virgin olive oil (VOO) at a proportion similar to hydroxytyrosol (HT) (160-479 micromol/kg oil). The present study was designed to measure the in vitro platelet antiaggregating activity of HT-AC in human whole blood, and compare this effect with that of HT and acetylsalicylic acid (ASA). The experiments were designed according to the standard procedure to investigate the activity of ASA. HT-AC and HT inhibited platelet aggregation induced by ADP, collagen or arachidonic acid in both whole blood and platelet-rich plasma (PRP). ASA and HT-AC had a greater effect in whole blood than in PRP when ADP or collagen was used as inducer. ASA and HT-AC had a greater effect in PRP+leucocytes than in PRP alone. All three compounds inhibited platelet thromboxane B2 and leucocyte 6-keto-prostaglandin F1alpha (6-keto-PF1 alpha) production. The thromboxane/6-keto-PGF1alpha inhibition ratio (as an indirect index of the prostanoid equilibrium) was 10.8 (SE 1) for HT-AC, 1.0 (SE 0.1) for HT and 3.3 (SE 0.2) for ASA. All three compounds stimulated nitric oxide production, although HT was a weaker effect. In our experiments only concentrations higher than 500 microm (HT) or 1 mm (HT-AC and ASA) inhibited 3-nitrotyrosine production. All three compounds inhibited the production of TNFalpha by leucocytes, with no significant differences between them. In quantitative terms HT-AC showed a greater antiplatelet aggregating activity than HT and a similar activity to that of ASA. This effect involved a decrease in platelet thromboxane synthesis and an increase in leucocyte nitric oxide production.

Lack of endothelium-derived hyperpolarizing factor (EDHF) up-regulation in endothelial dysfunction in aorta in diabetic rats.

It is not known whether the impairment of nitric oxide (NO)-dependent vasodilation of the aorta of diabetic rats is associated with any changes in the endothelial production of vasoactive prostanoids and endothelium-derived hyperpolarizing factor (EDHF). Therefore, we analyzed the contribution of NO, vasoactive prostanoids and EDHF to the decreased endothelium-dependent vasorelaxation in Sprague-Dawley rats at 4 and 8 weeks after diabetes mellitus induced by streptozotocin (STZ). The acetylcholine-induced (Ach) endothelium-dependent relaxation was significantly decreased in the thoracic aorta 8 weeks after the STZ-injection (Ach 10(-6) M: 73.1 +/- 7.4% and 56.7 +/- 7.9% for control and diabetic rats, respectively). The sodium nitroprusside-induced (NaNP) endothelium-independent vasodilation was also impaired in the diabetic rats (8 weeks after STZ) (NaNP 10(-8) M: 74.2 +/- 11.4% and 35.9 +/- 9.4% for control and diabetic rats, respectively). In contrast, the basal NO production, as assessed by the N omega-nitro-L-arginine methyl ester (L-NAME)-induced vasoconstriction was not modified in diabetes. Moreover, the amount of 6-keto-PGF(1 alpha) (stable metabolite of prostacyclin / prostaglandin I2 / PGI2 ), 12-L-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) and thromboxane B2 (TxB2 ) (stable metabolite of thromboxane A2 - TxA2) were significantly increased in the 8 weeks diabetic rat aorta. The EDHF-pathway did not change in the aortic endothelium during the development of STZ-induced diabetes. Our results indicate that STZ-induced diabetes mellitus did not modify the basal NO production, but induced the impairment of acetylcholine- and sodium nitroprusside-induced vasodilation in the thoracic aorta. In parallel with the impairment of NO-dependent vasodilation, the basal PGI2, 12-HHT and TxA2 synthesis were increased. The EDHF-pathway did not contribute to the endothelium-dependent relaxation either in control or diabetic aorta. The above alterations in the endothelial function may play an important role in the development of endothelial dysfunction and vascular complications of diabetes.

A role for cyclooxygenase in aging-related changes of beta-adrenoceptor-mediated relaxation in rat aortas.

beta-Adrenoceptor-mediated vasorelaxation decreases with age in various vascular beds. The present study investigated the roles of cyclooxygenase (COX) on beta-adrenoceptor vasorelaxation by isoprenaline in 8- and 54-week-old rat aortas. The vasorelaxation responses by isoprenaline (0.03-3 microM) were significantly reduced in 54-week-old aortas compared to 8-week. Addition of the non-selective COX inhibitors indomethacin (10 microM) or aspirin (10 microM) restored isoprenaline vasorelaxation of 54-week-old aortas to levels found in 8-week-old aortas. This suggests the involvement of COX prostanoids in the age-related reduction of beta-adrenoceptor vasorelaxation. Immunohistochemistry revealed greater levels of COX-1 and COX-2 staining in 54-week-old aortas compared to 8-week with expression located mainly in medial smooth muscle. An age-linked increase in COX-1 and COX-2 protein was found in cremaster arterioles of 54-week-old rats (compared to 8-week) mainly in the endothelial layer. The age-related increase in COX-1 and COX-2 protein led to elevation of prostacyclin (measured as 6-keto prostaglandin F(1alpha)) and thromboxane A(2) (measured as thromboxane B(2)) in 54-week compared to 8-week-old aortas. Endothelium removal in 54-week aortas markedly reduced the 6-keto prostaglandin F(1alpha) level, thus suggesting an endothelial source for elevated prostacyclin. These findings in combination with the effects of COX inhibitors suggest that the age-related decrease in beta-adrenoceptor vasorelaxation by isoprenaline is due to an age-linked increase in COX expression, which elevates production of COX-derived vasoactive prostanoids.

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells.

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1alpha, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.

Effect of chronic CB1 cannabinoid receptor antagonism on livers of rats with biliary cirrhosis.

Recent studies have shown that the activated endocannabinoid system participates in the increase in IHR (intrahepatic resistance) in cirrhosis. The increased hepatic production of vasoconstrictive eicosanoids is involved in the effect of endocannabinoids on the hepatic microcirculation in cirrhosis; however, the mechanisms of these effects are still unknown. The aim of the present study was to investigate the effects of chronic CB(1) (cannabinoid 1) receptor blockade in the hepatic microcirculation of CBL (common bile-duct-ligated) cirrhotic rats. After 1 week of treatment with AM251, a specific CB(1) receptor antagonist, IHR, SMA (superior mesenteric artery) blood flow and hepatic production of eicosanoids [TXB(2) (thromboxane B(2)), 6-keto PGF(1alpha) (prostaglandin F(1alpha)) and Cys-LTs (cysteinyl leukotrienes)] were measured. Additionally, the protein levels of hepatic COX (cyclo-oxygenase) isoforms, 5-LOX (5-lipoxygenase), CB(1) receptor, TGF-beta(1) (transforming growth factor beta(1)), cPLA(2) [cytosolic PLA(2) (phospholipase A(2))], sPLA(2) (secreted PLA(2)) and collagen deposition were also measured. In AM251-treated cirrhotic rats, a decrease in portal venous pressure was associated with the decrease in IHR and SMA blood flow. Additionally, the protein levels of hepatic CB(1) receptor, TGF-beta(1), cPLA(2) and hepatic collagen deposition, and the hepatic levels of 5-LOX and COX-2 and the corresponding production of TXB(2) and Cys-LTs in perfusates, were significantly decreased after 1 week of AM251 treatment in cirrhotic rats. Furthermore, acute infusion of AM251 resulted in a decrease in SMA blood flow and an increase in SMA resistance in CBL rats. In conclusion, the chronic effects of AM251 treatment on the intrahepatic microcirculation were, at least partly, mediated by the inhibition of hepatic TGF-beta(1) activity, which was associated with decreased hepatic collagen deposition and the activated PLA(2)/eicosanoid cascade in cirrhotic livers.

Endothelial cell COX-2 expression and activity in hypoxia.

The clinical experience with selective cyclooxygenase (COX)-2 inhibitors reveals there are important protective roles for COX-2 in the cardiovascular system. This study examined the response to hypoxia of endothelial cell eicosanoid synthesis with respect to the role of COX-2 and its molecular regulation in hypoxia. Human umbilical vein endothelial cells (HUVEC) were exposed to hypoxia and the effects on COX-2, prostacyclin (PGI(2)) and thromboxane (TXA(2)) synthesis were examined. COX-2 promoter constructs were used to examine the role of Hypoxia Inducible Factors (HIFs) in COX-2 responses to hypoxia. Hypoxia caused an increase in PGI(2) synthesis, but not TXA(2) synthesis. PGI(2), but not TXA(2) synthesis, was absolutely dependent on upregulation of COX-2 by hypoxia. Mutations of transcription factor binding sites in the promoter showed a lack of involvement of NFkappaB in the response to hypoxia, but suggested involvement of HIFs. Transfection of HUVEC with HIF expression vectors increased activity of the promoter construct and increased native COX-2 expression in normoxia. EMSA showed HIF binding in nuclear extracts of hypoxic HUVEC to a region of the COX-2 promoter. The endothelial cell response to hypoxia involves increased production of the anti-thrombotic eicosanoid, PGI(2), which is dependent on COX-2 upregulation by a HIF-mediated process.

Involvement of calcium/calmodulin-dependent protein kinase II to endotoxin-induced vascular hyporeactivity in rat superior mesenteric artery.

Endotoxin causes impaired vascular contractility proposed to be mediated mainly by induction of inducible nitric oxide synthase (iNOS). Evidence suggests that calcium/calmodulin dependent protein kinase II (CaMKII) may lead to activation of cytosolic phospholipase A(2alpha) (cPLA(2alpha))/inducible cyclooxygenase (COX-2) pathway in response to endotoxin in vascular smooth muscle cells. This study was conducted to determine if CaMKII is involved in the endotoxin-induced vascular hyporeactivity by activating of iNOS and/or cPLA(2alpha)/COX-2 enzymes in rat isolated superior mesenteric artery with endothelium. Incubation with endotoxin (100 microg ml(-1)) for 4h caused vascular hyporeactivity to norepinephrine which was completely abolished by phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3-PBIT), a selective iNOS inhibitor, methyl arachidonyl fluorophosphonate (MAFP), a selective 85kDa cPLA(2alpha) inhibitor, DFU, a selective COX-2 inhibitor, and KN-93, a selective CaMKII inhibitor. Endotoxin-induced increase in tissue nitrite production was decreased by 1,3-PBIT and DFU, and further increased by MAFP. MAFP, DFU and KN-93 reversed the endotoxin-induced decrease in tissue 6-keto-PGF(1alpha). These data suggest that reversal of the endotoxin-induced vascular hyporeactivity by inhibition of CaMKII in rat superior mesenteric artery may be related to increased production of vasodilator arachidonic acid products by cPLA(2alpha)/COX-2 pathway rather than prostacyclin and nitric oxide.

The involvement of a cyclooxygenase 1 gene-derived protein in the antinociceptive action of paracetamol in mice.

Paracetamol is a widely used analgesic and antipyretic with weak anti-inflammatory properties. Experimental evidence suggests that inhibition of prostaglandin biosynthesis contributes to its pharmacological actions. Three cyclooxygenase (COX) isoenzymes are involved in prostaglandin biosynthesis, COX-1, COX-2 and a recently discovered splice-variant of COX-1, COX-3. Our aim was to identify the relative roles for these enzymes in the antinociceptive action of paracetamol in mice. We compared the antinociceptive action of paracetamol with the non-selective non-steroid anti-inflammatory drug, diclofenac and studied paracetamol antinociception in COX-1 and COX-2 knockout mice. Paracetamol (100-400 mg/kg) inhibited both acetic acid- and iloprost-induced writhing responses. In contrast, diclofenac (10-100 mg/kg) inhibited only acetic acid-induced writhing. Only diclofenac reduced peripheral prostaglandin biosynthesis whereas both drugs reduced central prostaglandin production. Prostaglandin E(2) (PGE(2)) concentrations were reduced in different brain regions by administration of paracetamol. COX-1, COX-2 and COX-3 enzyme proteins were expressed in the same brain regions. The effects of paracetamol on writhing responses and on brain PGE(2) levels were reduced in COX-1, but not COX-2, knockout mice. The selective COX-3 inhibitors, aminopyrine and antipyrine also reduced writhing responses and brain PGE(2) biosynthesis. These results suggest that the antinociceptive action of paracetamol may be mediated by inhibition of COX-3.

Dalteparin, a low molecular weight heparin, attenuates inflammatory responses and reduces ischemia-reperfusion-induced liver injury in rats.

OBJECTIVE: To examine whether dalteparin, a low molecular weight heparin, prevents hepatic damage by inhibiting leukocyte activation, we analyzed its effect on ischemia/reperfusion (I/R) injury of rat liver in which activated leukocytes play a critical role. DESIGN: Prospective, randomized, controlled study. SETTING: Research laboratory at a university medical center. SUBJECTS: Male Wistar rats weighing 220-280 g. INTERVENTIONS: Hepatic damage was evaluated by changes in serum transaminase concentrations after I/R. Coagulation abnormalities were evaluated by changes in serum concentrations of fragment E of fibrin and fibrinogen degradation products after I/R. Hepatic tissue blood flow was measured by laser-Doppler flow meter. Hepatic edema was evaluated by determination of the change in the wet/dry tissue weight ratio. Rats were intravenously injected with dalteparin or unfractionated heparin (300 units/kg) and subcutaneously injected with DX9056a, a selective inhibitor of activated factor X (3 mg/kg). To determine whether dalteparin inhibits leukocyte activation, we examined the effect of dalteparin on hepatic concentrations of interleukin-12, tumor necrosis factor-alpha, and hepatic myeloperoxidase activity after I/R in vivo. In addition, we examined increases in tumor necrosis factor-alpha production in rat monocytes and in intracellular calcium concentrations in neutrophils in vitro. We also examined the effect of dalteparin on endothelial production of prostacyclin using isolated rat hepatic sinusoidal cells in vitro. MEASUREMENTS AND MAIN RESULTS: Intravenous administration of dalteparin inhibited increases in serum levels of both transaminases and serum concentrations of fragment E of fibrin and fibrinogen degradation products in animals subjected to hepatic I/R. Hepatic tissue blood flow after reperfusion was increased by dalteparin. Dalteparin inhibited hepatic edema, increases in hepatic tissue levels of interleukin-12 and tumor necrosis factor-alpha, and accumulation of neutrophils in animals subjected to hepatic I/R. Neither DX9065a nor unfractionated heparin showed any therapeutic effects, despite potent inhibition of increases in serum levels of fragment E of fibrin and fibrinogen degradation products. Neither monocytic tumor necrosis factor-alpha production nor neutrophil activation was inhibited by dalteparin in vitro. Dalteparin enhanced the hepatic I/R-induced increases in hepatic tissue levels of 6-keto-prostaglandin (PG) F1alpha, a stable metabolite of prostacyclin, which is capable of inhibiting monocytic tumor necrosis factor-alpha production. Pretreatment with indomethacin completely reversed both of the therapeutic effects of dalteparin, whereas pretreatment with NS-398, a selective inhibitor of cyclooxygenase-2, did not. Dalteparin did not directly increase the endothelial production of prostacyclin in vitro. CONCLUSION: Dalteparin might reduce I/R-induced liver injury in rats by attenuating inflammatory responses. These therapeutic effects might be independent of its anticoagulant activity but dependent on its capacity to enhance endothelial production of prostacyclin via cyclooxygenase-1 activation. Furthermore, the mechanism or mechanisms by which dalteparin promotes the endothelial production of prostacyclin in vivo might involve unknown factors other than endothelial cells.