Agomelatine inhibits platelet aggregation through melatonin receptor-dependent and independent mechanisms.

AIMS: Melatonin is known to inhibit platelet aggregation induced by arachidonic acid (AA). In the present study we investigated whether agomelatine (Ago), an antidepressant with agonist activity at melatonin receptor 1 (MT1) and MT2 could reduce platelets aggregation and adhesion. MAIN METHODS: Human platelets from healthy donors were used to test the in vitro effects of Ago in the presence of different platelet activators. We performed aggregation and adhesion assays, thromboxane B2 (TxB2), cAMP and cGMP measurements, intra-platelet calcium registration and flow cytometry assays. KEY FINDINGS: Our data revealed that different concentrations of Ago reduced AA- and collagen-induced human platelet aggregation in vitro. Ago also reduced AA-induced increase in thromboxane B2 (TxB2) production, intracellular calcium levels and P-selectin expression at plasma membrane. The effects of Ago in AA-activated platelets were likely dependent on MT1 as they were blocked by luzindole (a MT1/MT2 antagonist) and mimicked by the MT1 agonist UCM871 in a luzindole-sensitive manner. The MT2 agonist UCM924 was also able to inhibit platelet aggregation, but this response was not affected by luzindole. On the other hand, although UCM871 and UCM924 reduced collagen-induced platelet aggregation and adhesion, inhibition of collagen-induced platelet aggregation by Ago was not mediated by melatonin receptors because it was not affected by luzindole. SIGNIFICANCE: The present data show that Ago suppresses human platelet aggregation and suggest that this antidepressant may have the potential to prevent atherothrombotic ischemic events by reducing thrombus formation and vessel occlusion.

Maprotiline Ameliorates High Glucose-Induced Dysfunction in Renal Glomerular Endothelial Cells.

Maprotiline is an antidepressant that has been found to cause hypoglycemia. However, the effect of maprotiline on diabetic nephropathy (DN) has not been investigated. Here, we explored the effect of maprotiline on human renal glomerular endothelial cells (HRGECs) in response to high glucose (HG) stimulation. We found that maprotiline attenuated HG-induced oxidative stress in HRGECs with decreased reactive oxygen species production and increased superoxide dismutase activity. Maprotiline repressed the HG-induced expression of cyclooxygenases 2 at both mRNA and protein levels in HRGECs. The increased thromboxane B2 level and decreased 6-keto-prostaglandin F1α level induced by HG were significantly attenuated by maprotiline treatment. Maprotiline also prevented the HG-induced increase in the permeability of HRGECs and the decrease in the zonula occludens-1 expression and downregulated HG-induced increase in the expression of protein kinase C-α (PKC-α) in HRGECs. This protective effect of maprotiline on HG-induced HRGECs dysfunction was abolished by overexpression of PKC-α. In conclusion, maprotiline displayed a protective effect on HG-challenged HRGECs, which was mediated by the regulation of PKC-α. These findings provide further evidence for the potential use of maprotiline for the treatment of DN.

Urinary 11-dehydrothromboxane B2 aspirin efficacy testing is sensitive to perioperative inflammation in pediatric solid-organ transplant patients.

BACKGROUND: Evidence for aspirin efficacy testing in pediatrics is limited, especially outside of cardiology, yet thrombotic events have high morbidity in other areas such as pediatric transplant surgery. Debates about whether thromboembolic events while on aspirin represent "aspirin resistance" or "high on-treatment platelet reactivity" persist, given the poor intertest agreement between testing platforms. PROCEDURE: This prospective observational study involved measuring aspirin efficacy using ex vivo testing of platelet aggregation (VerifyNow-Aspirin, VN) and urine 11-dehydrothromboxane B2 (AsprinWorks, UTxB2) contemporaneously at up to three time points after major noncardiac organ transplant surgery. The collection days (CD) were the second and seventh days after stable aspirin dosing and then a convalescent time point 2-9 months later. RESULTS: Fifty-five participants (age range, 0-21 years) were enrolled, having undergone total pancreatectomy with islet autotransplantation (N = 36), orthotopic liver transplantation (N = 18), and combined liver-kidney transplantation (N = 1). Platelet reactivity measured by VN remained unchanged, whereas UTxB2, which was elevated postoperatively, decreased significantly from CD1 to CD2 and CD3. Discordance in therapeutic efficacy was noted per manufacturer cutoffs, with therapeutic VN results in 86% of tests, whereas 12% of UTxB2 were therapeutic. Age-based stratification of UTxB2 results using previously published pediatric median levels increased overall UTxB2 therapeutic rates (80%) and intertest concordance (67% vs 27% if using adult range). No thrombotic events were observed. CONCLUSIONS: Our data suggest that urine thromboxane production may be an underappreciated reflection of postoperative inflammation. Validation of pediatric normal ranges for UTxB2 is a critical next step.

Evaluation of the antithrombotic activity of Zhi-Xiong Capsules, a Traditional Chinese Medicinal formula, via the pathway of anti-coagulation, anti-platelet activation and anti-fibrinolysis.

Zhi-Xiong Capsules (ZXC) involving Hirudo, Ligusticum chuanxiong, Salvia miltiorrhiza, Leonurus artemisia, and Pueraria lobata, is an empirical prescription used in Chinese clinics applied for treating cerebral arteriosclerosis and blood-stasis in clinic. However, the mechanism of its antithrombotic activity has not been investigated until now. The present study was designed to investigate its antithrombotic effects, the mechanism of ZXC on anti-thrombus action and to identify the main chemical composition of ZXC using HPLC-DAD-ESI-IT-TOF-MS. Two animal models were used to evaluate the antithrombotic effect of ZXC, the arterial thrombosis model and a venous thrombosis model. ZXC prolonged the plasma recalcification time (PRT), the activated partial thromboplastin time (APTT), the thrombin time (TT) and the prothrombin time (PT) and clearly reduced the content of fibrinogen (FIB) obviously in the arterial thrombosis model. Furthermore, it markedly suppressed the level of TXB2 and up-regulated the level of 6-keto-PGF1a. In addition, it significantly up-regulated the level of t-PA and down-regulated the level of PAI-1 (p < 0.05). These results revealed that ZXC played a vital role in the prevention of thrombosis through interacting with multiple targets, including inhibition of coagulation and platelet aggregation and increasing thrombolysis. A total of 23 compounds were identified as the main components of ZXC by HPLC-DAD-ESI-IT TOF-MS.

Up-regulation of Kv7.1 channels in thromboxane A2-induced colonic cancer cell proliferation.

Thromboxane A2 (TXA2) is known to stimulate colonic cancer cell proliferation, although the mechanism has not been clarified. In this study, we compared the expression levels of Kv7.1 K(+) channels between human colorectal cancer tissue and the accompanying non-tumor mucosa. Kv7.1 proteins were found to be consistently up-regulated in the cancer tissues from different patients. Kv7.1 was also expressed in human colonic cancer cell lines. Treatment of colonic cancer cells with 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of TXA2, significantly increased whole-cell K(+) currents sensitive to chromanol 293B, an inhibitor of Kv7.1 channels, in parallel with an increased expression of Kv7.1 proteins. In contrast, TXB2, an inactive metabolite of TXA2, had no effects on expression level and function of Kv7.1. A TXA2 receptor antagonist (SQ29548) and an inhibitor of cAMP-dependent protein kinase (Rp-8-Br-MB-cAMPS) inhibited STA2-induced increases in both Kv7.1 expression and chromanol 293B-sensitive K(+) currents. Interestingly, STA2-stimulated proliferation of colonic cancer cells was inhibited by chromanol 293B. These results suggest that Kv7.1 channels are involved in the TXA2-induced cancer cell proliferation and that they are up-regulated by the TXA2 receptor-mediated cAMP pathway.

Select dietary phytochemicals function as inhibitors of COX-1 but not COX-2.

Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors. Many active dietary factors are reported to suppress carcinogenesis by targeting COX-2. A major question was accordingly raised: why has the lifelong use of phytochemicals that likely inhibit COX-2 presumably not been associated with adverse cardiovascular side effects. To answer this question, we selected a library of dietary-derived phytochemicals and evaluated their potential cardiovascular toxicity in human umbilical vein endothelial cells. Our data indicated that the possibility of cardiovascular toxicity of these dietary phytochemicals was low. Further mechanistic studies revealed that the actions of these phytochemicals were similar to aspirin in that they mainly inhibited COX-1 rather than COX-2, especially at low doses.

Vasoconstrictor potential of coronary aspirate from patients undergoing stenting of saphenous vein aortocoronary bypass grafts and its pharmacological attenuation.

RATIONALE: Stent implantation into atherosclerotic plaques releases, apart from particulate debris, soluble substances that contribute to impaired microvascular perfusion. OBJECTIVE: To quantify the release of vasoconstrictors and to determine the efficacy of coronary dilators to attenuate their action. METHODS AND RESULTS: Using a distal protection/aspiration device, coronary arterial blood was retrieved before and during stenting in 22 patients with severe saphenous vein aorto-coronary bypass stenoses. The release of catecholamines, endothelin, serotonin, thromboxane B(2), and tumor necrosis factor (TNF)α was measured. The response of rat mesenteric arteries with intact (+E) and denuded (-E) endothelium to aspirate plasma was normalized to that by KCl. Responses to selective receptor blockade, adenosine, nitroprusside, and verapamil against the aspirate-induced constriction were determined. The coronary arterial plasma withdrawn before stenting induced 21±5% and the aspirate plasma after stenting induced 95±8% of maximum KCl-induced vasoconstriction. Serotonin, thromboxane B(2), and TNFα release into aspirate plasma increased by 1.9±0.2 µmol/L, 25.6±3.1 pg/mL, and 19.7±6.1 pg/mL, respectively, during stenting. The aspirate-induced vasoconstriction was largely antagonized by selective serotonin receptor blockade, with little further antagonism by additional thromboxane receptor blockade. TNFα did not induce constriction per se but potentiated the constriction with serotonin and the thromboxane-analog U-46619 in arteries +E. The concentrations to induce half-maximal vasodilation were comparable for nitroprusside (+E, 3.3×10(-8); -E, 1.9×10(-8) mol/L) and verapamil (+E, 8.3×10(-8); -E, 7.8×10(-8) mol/L), and the vasoconstriction was eventually eliminated. The vasodilator response to adenosine was dependent on functional endothelium and weaker. CONCLUSION: Serotonin is the main coronary vasoconstrictor after stenting, and thromboxane and TNFα somewhat potentiate the serotonin response. Nitroprusside and verapamil are more potent than adenosine to attenuate the aspirate plasma-induced vasoconstriction, and they are not dependent on functional endothelium.

Effects of aspirin and NO-aspirin (NCX 4016) on platelet function and coagulation in human endotoxemia.

Acetylsalicylic acid (ASA) prevents thromboembolic events by inhibiting platelet function through blocking of cyclooxygenase type 1 (COX-1). A nitroderivate of ASA, 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)-phenyl ester (NCX 4016) was synthesized, which additionally acts through nitric oxide release. In various in vitro and animal studies NCX 4016 exhibited antithrombotic and anti-platelet properties. We used the standardized model of endotoxin infusion into human volunteers to compare the effects of NCX 4016 and ASA on platelet function and TF-induced coagulation activation. The trial consisted of two parts. In the first part, 10 healthy male volunteers were included in a randomized, open cross-over trial to find a NCX formulation with optimal tolerability and pharmacokinetic data were obtained. The second part was a randomized, double blind placebo controlled clinical trial consisting of 30 healthy male volunteers in three parallel groups (n = 10 per group). Volunteers received either NCX 4016 (800 mg b.i.d.), ASA (425 mg b.i.d.) or placebo for 7 days, before infusion of 2 ng/kg endotoxin on day 8. ASA attenuated the endotoxin-induced platelet plug formation (measured by PFA-100) significantly better than NCX 4016 and placebo (p < 0.004), while there was no difference in soluble P-selectin or VWF-levels. Urine 11-dehydro-thromboxane B(2) levels were significantly lower in the ASA and NCX 4016 groups as compared to placebo (p < 0.05). Neither ASA nor NCX 4016 significantly changed prothrombin fragment(1 + 2), D-Dimer or tissue factor (TF)-mRNA levels. In summary, NCX 4016 had no effect on VWF release, platelet activation as measured by soluble P-selectin or TF gene expression. NCX 4016, at the dose tested, unlike ASA, had no effect on platelet collagen/epinephrine induced plug formation under high shear rates.

The effect of dobutamine on platelet aggregatory function in newborn piglets with hypoxia and reoxygenation.

Dobutamine, a beta-adrenoceptor agonist that is often used to treat myocardial dysfunction in asphyxiated neonates, may act on the adrenoceptors of platelets resulting in activation. Little information is available on the effect and mechanistic pathway of dobutamine on the platelet aggregatory function in neonatal asphyxia. Newborn piglets were acutely instrumented and exposed to hypoxia for 2 h and reoxygenation for 4 h. Piglets were randomized to receive dobutamine infusion (5, 10, or 20 microg/kg per min) or saline (hypoxic-control) at 2 to 4 h of reoxygenation (n = 8 each), and sham-operated animals were not exposed to hypoxia and reoxygenation (n = 6). Platelet number, collagen-stimulated whole blood aggregation, and plasma concentrations of thromboxane B2 were studied. The effects of alpha- and beta-adrenoceptor antagonists (phentolamine and propranolol, respectively) on platelet aggregation to in vitro administration of dobutamine (3 microM) were also examined. Shock and metabolic acidosis developed similarly in all hypoxia-reoxygenated groups. At 4 h of reoxygenation, platelet numbers in all groups decreased, with no differences among groups. Platelet aggregation deteriorated significantly with a rightward shift of concentration-response curve in piglets receiving 10 and 20 microg/kg per min of dobutamine. The group that received 20 microg/kg per min of dobutamine had increased plasma thromboxane B2 concentrations from baseline (P < 0.05). The platelet aggregatory response induced by 3 microM of dobutamine was improved by the coadministration of the beta-but not the alpha-adrenoceptor antagonist. We observed platelet aggregatory dysfunction in hypoxic-reoxygenated newborn piglets treated with high-dose dobutamine. Further investigation is needed to examine the differential effects of dobutamine and hypoxia-reoxygenation in platelet aggregation in newborns.

Role of eicosanoids of the contralateral kidney in maintenance of two-kidney, one-clip renovascular hypertension in rats.

OBJECTIVE: To elucidate the role of the eicosanoids prostaglandin E(2) (PGE(2)), 6-keto-prostaglandin F(1a) (PGF(1a)) and thromboxane B(2) (TXB(2)) in the maintenance of two-kidney, one-clip renovascular hypertension in rats. MATERIAL AND METHODS: The right renal artery was constricted by a silver clip in 63 male Sprague-Dawley rats to induce hypertension, while a sham operation was performed in 17 control rats. Six months after the induction of hypertension, nephrectomy of the clipped kidney was performed. Nephrectomy was followed by a period of high sodium intake. Blood pressure and eicosanoid excretion were measured before and after nephrectomy of the clipped kidney, as well as during high sodium intake. RESULTS: During the chronic phase of Goldblatt hypertension, the amount of vasoconstrictive TXB(2) excreted by the contralateral kidney increased compared to that in the controls, whereas PGE(2) excretion was unaffected. Eicosanoid excretion before and after removal of the clipped kidney did not differ between post-Goldblatt hypertensive and post-Goldblatt normotensive animals. During the period of high sodium intake, PGE(2) excretion increased only in control rats, being unaltered in Goldblatt hypertensive rats. CONCLUSIONS: In the chronic phase of two-kidney, one-clip renovascular hypertension, the contralateral kidney of post-Goldblatt hypertensive and post-Goldblatt normotensive rats excretes more vasoconstrictive thromboxane in comparison to controls, whereas excretion of vasodilatory prostaglandin is not elevated. However, increased TXB(2) excretion and the absence of an increase in PGE(2) excretion from the contralateral kidney do not appear to be important for the maintenance of high blood pressure in this model of renovascular hypertension.

[Effect of thromboxane B2 on morphological features of apoptosis in cultured rat hepatocytes exposed to hepatotropic substances].

Thromboxanes (TX) are known to have damaging effect on a liver but their influence on the cell death, in particular on hepatocyte apoptosis and its morphological features is not investigated enough. Cell death of the rat hepatocytes was investigated in primary culture by double vital staining with fluorescent nuclear stains Hoechst 33342 and propidium iodide and by electron microscopy. It was established that exogenous Tx B2 increases the amount of hepatocytes with early stages of apoptosis - the condensed chromatin and nucleus and cell size reduction. The changes in a percentage of hepatocytes with morphological features of the late stages of apoptosis - fragmented nuclei and division on apoptotic bodies were not revealed. Tx B2 intensified the carbon tetrachloride action on hepatocyte apoptotic death and increased chromatin condensation. Tx B2 application to hepatocytes injured by chenodeoxycholic acid significantly increased the amount of cells with a final stage ofapoptosis.

Polymorphisms of COX-1 and GPVI associate with the antiplatelet effect of aspirin in coronary artery disease patients.

The antiplatelet effect of aspirin varies individually. This study evaluated whether the antiplatelet effect of aspirin associates with polymorphisms in the genes coding for cyclo-oxygenase-1 (COX-1) and several platelet glycoprotein (GP) receptors in patients with stable coronary artery disease (CAD). Blood samples were collected from 101 aspirin-treated (mean 100 mg/d) patients. Compliance to treatment was assessed by plasma salicylate measurement. Platelet functions were assessed by two methods: 1) Response to arachidonic acid (AA, 1.5 mmol/L in aggregometry, and 2) PFA-100, evaluating platelet activation under high shear stress in the presence of collagen and epinephrine (CEPI). Aspirin non-response was defined as: 1) slope steeper than 12%/min in AA-aggregations, and 2) by closure time shorter than 170 s in PFA-100. The methods used detected different individuals as being aspirin non-responders. Five and 21 patients, respectively, were non-responders according to AA-induced aggregation and PFA-100. Increased plasma thromboxane B2 levels correlated with poor aspirin-response measured with both AA-induced aggregations and PFA-100 (P = 0.02 and P = 0.003, respectively). Of the non-responders detected by AA, 3 of 5 (60%) carried the rare G allele for the -A842G polymorphism of COX-1 in contrast to 16 of 96 (17%) responders (P = 0.016). Diabetes was associated with poor response. Aspirin non-response detected by PFA-100 associated with C13254T polymorphism of GP VI and female gender (P = 0.012 and P = 0.019, respectively). Although two patients were possibly non-compliant, this did not effect present conclusions. Evaluation of aspirin efficacy by AA-induced aggregation and PFA-100 detected different individuals, with different genotypic profiles, as being aspirin non-responders.

Effect of prostaglandins against alloxan-induced diabetes mellitus.

Previously, we observed that alloxan-induced in vitro cytotoxicity and apoptosis in an insulin secreting rat insulinoma, RIN, cells was prevented by prior exposure to prostaglandin (PG) E(1), PGE(2), PGI(2), PGF(1)(alpha), and PGF(3)(alpha) (P<0.05 compared to alloxan), whereas thromboxane B(2) (TXB(2)) and 6-keto-PGF(1)(alpha) were ineffective. In an extension of these studies, we now report that prior intraperitoneal administration of PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) prevented alloxan-induced diabetes mellitus in male Wistar rats, whereas PGI(2), TXB(2), and 6-keto PGF(1)(alpha) were not that effective. PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) not only attenuated chemical-induced diabetes mellitus but also restored the antioxidant status to normal range in red blood cells and pancreas. These results suggest that PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) can abrogate chemically induced diabetes mellitus in experimental animals and attenuate the oxidant stress that occurs in diabetes mellitus.

Can platelet function tests predict the clinical efficacy of aspirin?

"Aspirin resistance" and "aspirin nonresponsiveness" are terms used both to describe both the failure of aspirin to protect subgroups of individuals from severe vascular events and to evoke an appropriate inhibition of platelet function. Several studies utilizing a broad range of platelet function tests have shown that some subgroups of individuals exhibit a reduced or completely missing antiplatelet response to aspirin. The clinical significance of aspirin nonresponsiveness for the prediction of clinical endpoints remains, however, to be determined. Thus far, only three prospective clinical trials have demonstrated a possible relationship between aspirin nonresponsiveness and subsequent vascular events. Most platelet function tests used in respective clinical studies cannot be reliably performed in clinical routine and are not interchangeable for monitoring antiplatelet treatment. There is a need for a simple and reliable assay for predicting the clinical efficacy of antiplatelet therapy. Recent data demonstrate that none of the currently developed assays, including the PFA-100 system, are presently able to accomplish these objectives.

Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages.

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.

Biological assessment of aspirin efficacy on healthy individuals: heterogeneous response or aspirin failure?

BACKGROUND AND PURPOSE: The widespread use of aspirin requires clarification of the aspirin resistance phenomenon. Most studies on this field are focused on patients which may affect the action of aspirin. METHODS: We evaluated the biological efficacy of aspirin in healthy subjects. RESULTS: Agonist-induced platelet aggregation was fully abrogated by 100 mg of aspirin in all individuals. By contrast, with the platelet function analyzer-100 device, 33.3% of the subjects displayed no response. This failure was overcome by 500 mg or by in vitro treatment of blood with 30 mumol/L acetylsalicylic acid. Intake of 100 mg of aspirin efficiently reduced by 75% the level of 11-dehydro thromboxane B2 (11-dTxB2) in all cases. However, variability on the pre-aspirin level (range 72.4 to 625.9 ng/mmol creatinine) led to substantial differences in the residual amount of the metabolite between subjects treated with aspirin (range 12.9 to 118.0 ng/mmol creatinine). Finally, there was no influence of platelet glycoprotein IIb/IIIa (Pro33Leu), platelet glycoprotein Ia/IIa, (C807T), and FXIII (Val34Leu) polymorphisms on the efficacy of aspirin. However, the cyclooxygenase (Cox)-1 50T allele associated with higher level of 11-dTxB2, both before and after aspirin. Moreover, the Cox-2 -765C variant displayed a slightly higher reduction in 11-dTxB2 level on treatment with aspirin. CONCLUSIONS: Our findings suggest that full resistance of healthy subjects to aspirin is rather unlikely. However, differences in aspirin absorption, or pharmacokinetic, or other unrecognized factors may lead to lack of effect of low dose of aspirin in some subjects when using tests like platelet function analyzer-100. Whether Cox polymorphisms are thrombotic risk factor for patients under aspirin will require further research.

Regulation of Candida albicans morphogenesis by fatty acid metabolites.

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. C. albicans morphogenesis is regulated by multiple signals and signaling pathways. However, signals that control morphogenesis in vivo are unknown. We investigated the effects of host long chain fatty acids, eicosanoids, and bacterial short chain fatty acids on control of germination. None of the C18 or C20 fatty acids tested had an effect on enhancing germ tube formation (arachidonic acid, oleic acid, linolenic acid, or gamma-linolenic acid). Among the different eicosanoids, both prostaglandin E2 and thromboxane B2 significantly enhanced serum-induced germination by C. albicans. Addition of antiprostaglandin or antithromboxane antibodies to serum alone inhibited germ tube formation by almost 30%, while control antibody had no effect, indicating that these eicosanoids are major morphogenic factors in the serum. Since these molecules also bind to albumin, this may also explain the hyphal transforming activity in serum that associates with albumin. Interestingly, short chain fatty acids (butyric acid), the product of lactic acid bacteria (LAB), inhibited germination. In addition, LAB culture supernatants as well as live LAB also inhibited C. albicans morphogenesis. Overall, these results indicate that fatty acid metabolites and fatty acid pathways can up-regulate and down-regulate germination in C. albicans.

Effect of prostaglandins against alloxan-induced cytotoxicity to insulin secreting insulinoma RIN cells in vitro.

In the present study, we studied the effect of various prostaglandins (PGs) on alloxan-induced cytotoxicity to rat insulinoma (RIN) cells. Of all the PGs tested, PGE(1), PGE(2), PGI(2), PGF(1 alpha), and PGF(3 alpha) protected RIN cells from alloxan-induced cytotoxicity (P<0.05 compared to alloxan), whereas thromboxane B(2) and 6-keto-PGF(1 alpha) were not effective. PGE(1) induces a statistically significant increase in the activities of superoxide dismutase and glutathione peroxidase and decrease in lipid peroxides in alloxan-treated RIN cells (P<0.001). PGE(1) restored nitric oxide/lipid peroxide ratio to normalcy, suggesting that PGE(1) suppresses oxidant stress induced by alloxan in RIN cells in vitro. Furthermore, PGE(1) prevented DNA damage and apoptosis induced by alloxan. These results indicate that PGE(1) prevents alloxan-induced cytotoxicity to RIN cells in vitro.

Cyclooxygenase inhibition is associated with downregulation of apolipoprotein AI promoter activity in cultured hepatoma cell line HepG2.

Prostanoids have been implicated in the transcriptional control of several genes. Since prostanoid synthesis inhibitors are commonly used in subjects with coronary heart disease we studied the effect of cyclooxygenase (COX) inhibition on apolipoprotein AI (apoAI) expression in a human hepatoma cell line (HepG2) transfected with full-length apoAI promoter attached to the chloramphenicol acetyl transferase (CAT) reporter gene. To control for transfection efficiency, the cells were cotransfected with the plasmid pCMV.SPORT-beta-gal containing the beta-galactosidase gene driven by the cytomegalovirus promoter. Treatment of these cells with varying concentrations of indomethacin (INDO, 0, 50, 100, and 300 micromol/L) resulted in a dose-dependent decrease in apoAI promoter activity (% acetylation corrected for beta-galactosidase activity: were 46.1 +/- 2.6, 29.9 +/- 1.2, 25.2 +/- 2.9, and 17.2 +/- 2.8, respectively, P <.001). INDO treatment did not cause significant changes in beta-galactosidase activity. A similar reduction in apoAI promoter activity was found after treating the cells with 50 micromol/L acetylsalicylic acid (ASA) (31.8 +/- 1.8%, P <.001), suggesting that the effect of INDO is related to COX inhibition rather than a peculiar effect of INDO. Nuclear run-off assays indicated that treatment of cells with 50 micromol/L INDO resulted in 31.4% reduction in apo A1 transcription rate (P <.0002). Northern blot analysis of RNA from HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed that the apoAI mRNA concentration relative to G3PDH mRNA was 4,043.0 +/- 84.6 and 3,064.0 +/- 49.8 in control and INDO-treated cells, respectively (P <.0006). Kinetic studies of apoAI mRNA in HepG2 cells indicated that the half-life of apoAI mRNA was not significantly altered with 50 micromol/L INDO treatment. Apo AI mRNA half-life was 25.3 hours in control cells and 26.9 hours in INDO-treated cells. Western blot analysis of culture media of HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed a significant reduction in apoAI protein (6,760.0 +/- 318.1 v 4,773.0 +/- 112.0 arbitrary units, P <.004). Treatment of cells with either arachidonic acid (COX substrate) or various prostanoids including prostaglandin I(2), thromboxane B(2), (+/-)5-HETE, or (+/-)12-HETE did not significantly alter apoAI promoter activity. However, prostaglandin E(1) and E(2) at the highest concentration tested (50 nmol/L) significantly repressed apoAI promoter activity. COX activity measurements in HepG2 cells verified the efficacy of COX inhibition by INDO. It is concluded that COX inhibition with INDO or ASA downregulates apoAI expression at the transcriptional level. This effect could not be attributed to either arachidonic acid excess or to a deficiency in various prostanoids tested.

11-Dehydro-thromboxane B2, a stable thromboxane metabolite, is a full agonist of chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) in human eosinophils and basophils.

Thromboxane (TX) A(2), a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB(2), which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB(2), but not the TXA(2) analogue U46,619 or TXB(2), activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB(2) was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D(2) has been shown to be its principal ligand. 11-Dehydro-TXB(2) induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD(2) but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB(2) were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB(2)- and PGD(2)-induced shape change. 11-Dehydro-TXB(2) also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB(2) had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB(2) is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB(2)/CRTH2axis may be of therapeutic relevance.