Genistein Ameliorates Non-alcoholic Fatty Liver Disease by Targeting the Thromboxane A2 Pathway.

Non-alcoholic fatty liver disease (NAFLD) is now a public health issue worldwide, but no drug has yet received approval. Genistein, an isoflavonoid derived from soybean, ameliorates high-fat-diet-induced NAFLD in mice, but the molecular underpinnings remain largely elusive. Arachidonic acid (AA) is a major ingredient of animal fats, and the AA cascade has been implicated in chronic inflammation. In this study, we investigated whether genistein was against NAFLD by targeting the AA cascade. Using a mouse model, we showed that genistein supplementation improved high-fat-diet-induced NAFLD by normalizing hepatomegaly, liver steatosis, aminotransferase abnormalities, and glucose tolerance. The thromboxane A2 (TXA2) pathway was aberrantly active in NAFLD, evidenced by an elevation of circulating TXA2 and hepatic thromboxane A2 receptor expression. Mechanistically, we found that genistein directly targeted cyclooxygenase-1 activity as well as its downstream TXA2 biosynthesis, while the TXA2 pathway might mediate NAFLD progression by impairing insulin sensitivity. Taken together, our study revealed a crucial pathophysiological role of the TXA2 pathway in NAFLD and provided an explanation as to how genistein was against NAFLD progression.

Amiloride reduces portal hypertension in rat liver cirrhosis.

OBJECTIVE: This study aimed to investigate the effect of amiloride on portal hypertension. Amiloride is known to inhibit Na(+)/H(+) exchangers on activated hepatic stellate cells. METHODS: Liver cirrhosis in rats was induced by bile duct ligation (BDL) or thioacetamide (TAA) administration. The effects of zymosan for Kupffer cell (KC) activation or a thromboxane (TX) analogue (U46619) were tested in isolated perfused livers of cirrhotic rats and in vivo. Downstream mechanisms were investigated using Rho kinase inhibitor (Y-27632) or amiloride. Acute and chronic effects of amiloride and canrenoate on portal pressure were compared in perfused livers and in vivo. TXB(2) efflux was measured by ELISA. The phosphorylation state of moesin (p-moesin) as an indicator of Rho kinase activity and expression of the thromboxane synthase were assessed by western blot analyses. The activity of hepatic stellate cells was analysed by western blot and staining for alpha-smooth muscle actin (alpha-SMA). RESULTS: In BDL rats, KC activation via zymosan increased portal pressure. This was attenuated by the Rho kinase inhibitor Y-27632. Increased thromboxane efflux following zymosan infusion remained unaltered by Y-27632. The infusion of amiloride attenuated zymosan- and U46619-induced increases in portal perfusion pressure. In vivo, direct administration of amiloride, but not of canrenoate, lowered portal pressure. In TAA and BDL rats, treatment with amiloride for 3 days reduced basal portal pressure and KC-induced increases in portal pressure whereas canrenoate had no effect. In livers of amiloride-treated animals, the phosphorylation state of moesin and the number of alpha-SMA positive cells were reduced. CONCLUSIONS: Amiloride lowers portal pressure in rat liver cirrhosis by inhibition of intrahepatic vasocontraction. Therefore, patients with cirrhosis and portal hypertension may benefit from amiloride therapy.

[Terutroban and endothelial TP receptors in atherogenesis]. / Terutroban et récepteurs TP endothéliaux dans l'athérogenèse.

Treatment of thrombotic diseases implicates the use of anti-platelet agents, anti-coagulants and pro-fibrinolytic substances. Amongst the anti-platelet drugs, aspirin occupies a unique position. As soon as it became evident that the major action of aspirin is indirect blockade, through inhibition of cyclooxygenase (COX), of the production of thromboxane A2 (TXA2), a powerful vasoconstrictor and platelet activator, research for new anti-thrombotics that interact more specifically with the production and/or the action of TXA2 was started. Terutroban (S 18886) is a selective antagonist of TP receptors, the receptors for TXA2, that are present on platelets and on vascular smooth muscle cells, but also on endothelial cells. The role played by the platelet and smooth muscle cell TP receptors in thrombotic disease is well known, and preclinical and clinical studies with terutroban have illustrated the powerful antithrombotic effects of this agent. The implication of endothelial TP receptors in the development of atherosclerotic disease has only been examined during the past five years and studies with terutroban have been crucial for understanding the role of these endothelial receptors in cardiovascular physiopathology. The goal of the present review is to discuss the arguments in favour of the hypothesis suggesting that activation of endothelial TP receptors, by causing expression of adhesion molecules, favours adhesion and infiltration of monocytes/macrophages in the arterial wall, thereby stimulating the development of atherosclerosis. The review will also highlight the important contribution of the studies performed with terutroban in this research area. The triple activity (anti-thrombotic, anti-vasoconstrictor, anti-atherosclerotic) observed with terutroban in preclinical studies, stressed by the first results in clinical development, places terutroban as an innovative drug with a unique potential for treatment of cardiovascular disorders.

Pathogenesis of a bleeding disorder characterized by platelet unresponsiveness to thromboxane A2.

A platelet disorder characterized by the absence of thromboxane A2 (TXA2)-induced platelet aggregation is a new clinical entity of platelet dysfunction. The platelets of three patients had the ability to bind exogenous TXA2, but synthetic TXA2 mimetic-induced postreceptor biochemical events, such as IP3 formation, Ca2+ mobilization, phosphatidic acid formation, and GTPase activities, were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. Gene analysis of the TXA2 receptor showed a substitution of Leu for Arg60 in the first cytoplasmic loop in all patients, and this mutation seemed to be responsible for this platelet disorder.

[Effect of losartan on human platelet activation by thromboxane A2]. / Efecto del losartán sobre la activación de plaquetas humanaspor tromboxano A2.

INTRODUCTION AND OBJECTIVES: Previous studies have demonstrated that losartan, an AT-1 receptor antagonist of angiotensin II (Ang II) could block the receptor of thromboxane A2 (TXA2) in the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. MATERIALS AND METHODS: Platelets were obtained from 15 healthy men between the age 26 and 40. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by a synthetic TXA2 analogue, U46619 (5 x 10(-6) mol/l). RESULTS: The U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-response manner. Only a high dose of EXP 3174 (5 10-5 mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I-converting inhibitor failed to modify U46619-induced platelet aggregation. Despite the platelets expressing AT-1 type receptors, of Ang II exogenous Ang II did not modify platelet aggregation induced by U46619. The binding of U46619 to platelets was competitively inhibited by losartan in dose-dependent manner. However, only a high dose of EXP 3174 reduced the binding of U46619. Captopril failed to modify the binding of U46619 to platelets. CONCLUSIONS: Losartan decreased platelet aggregation by a TXA2-dependent mechanism. EXP 3174 showed a lesser potency than losartan to reduce TXA2-platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced TXA2-dependent platelet activation independently of the blockade of AT-1 receptors.

Eicosapentaenoic acid suppresses PDGF-induced DNA synthesis in rat mesangial cells: involvement of thromboxane A2.

BACKGROUND: The administration of eicosapentaenoic acid (EPA) derived from marine oils has been shown to suppress vascular myocyte, lymphocyte, keratinocyte, and mesangial cell proliferation in vitro, although the effects are variable and most reports provide fragmented insight into the mechanism(s) responsible for altering cell growth, particularly the relationship of membrane lipid remodeling to changes in cell proliferation. Thus, these studies were designed to elucidate the effects of mesangial cell membrane fatty acid remodeling (induced by EPA) on cell growth, and to define the relevance of changes in the synthesis of growth-modulating eicosanoids. METHODS: Mesangial cells were grown in RPMI and 17% fetal calf serum, and were subcultured and grown for 48 hours in 17% delipidated serum or delipidated serum supplemented with 0 to 50 micrograms/mL of EPA. Quiescent EPA-loaded and control mesangial cells were subjected to stimulation with 20 ng/mL of platelet-derived growth factor (PDGF) followed by measurement of 3H-thymidine incorporation and cell number. RESULTS: Mesangial cells remodeled with EPA exhibited a significant decrease in PDGF-stimulated 3H-thymidine incorporation and cell number associated with a reduction in thromboxane A2 (TXA2) in the media. Importantly, the phospholipid fatty acid composition of mesangial cells grown in media enriched with EPA revealed an increase in EPA (0.5 +/- 0.02% to 17.02 +/- 0.52%) coupled with a reciprocal decrease in the precursor for TXA2, arachidonic acid (18.9 +/- 3.17% to 3.55 +/- 0.30%). Blockade of TXA2 synthesis in mesangial cells treated with indomethacin (0.1 to 100 mumol/L) or the specific TXA2 synthase inhibitor, U-63557A (0.1 to 100 mumol/L), evoked a similar reduction in PDGF-stimulated proliferation and TXA2 synthesis. Coincubation of PDGF with the TXA2 mimetic, U-46619 (1 mumol/L), reversed the growth suppression induced by cell membrane remodeling. CONCLUSIONS: These studies suggest that changes in membrane fatty acid composition induced by EPA modulates PDGF-stimulated proliferation by engendering a change in PDGF-stimulated TXA2 synthesis. Furthermore, we conclude that TXA2 functions as a comitogen for PDGF-stimulated mesangial cell growth.

Interaction of acetylcholine and endothelin-1 in the modulation of pulmonary arterial pressure.

OBJECTIVE: The study was designed to investigate the effects of acetylcholine (ACh) on pulmonary circulation with special regard to mediators that could be involved in the mediation of ACh-induced effects. ACh has been reported to induce either vasodilation or vasoconstriction in the pulmonary circulation of different species. DESIGN: Prospective experimental study in rabbits. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Sixty-six adult rabbits of either sex. INTERVENTIONS: The experiments were performed on 66 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. ACh was injected in various concentrations after pulmonary artery preconstriction and in untreated lungs. MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. Perfusate samples were taken intermittently to determine endothelin-1 (ET-1), thromboxane A2 (TXA2), and prostacyclin (PGI2) concentrations. ACh in final dosages from 10(-5) to 10(-2) M (n = 6 each) was injected into the pulmonary artery of lungs treated with U46619 to induce pulmonary arterial hypertension or was injected into untreated lungs. To analyze the potential mechanisms of action, ACh (10(-5) M) was administered in additional experiments after pretreatment with either ETA receptor antagonist BQ123 (10(-6) M; n = 6) or the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6). In preconstricted pulmonary vessels, ACh (10(-3) and 10(-2) M) initially induced a PAP rise for 10 mins followed by a sustained decrease. In untreated lungs, ACh induced an immediate dose-dependent increase in PAP, requiring as long as 30 mins to return to predrug levels. Simultaneously, significantly elevated TXA2 and PGI2 levels were observed. Furthermore, ET-1 was detected in the perfusate, which was free from ET-1 before ACh administration. Pretreatment with BQ123 reduced substantially the ACh (10(-5) M)-induced PAP increase and the release of TXA2 and PGI2. At 5 mins, the PAP maximum was reduced from 18.5 +/- 3.2 mm Hg to 9.9 +/- 0.65 mm Hg by BQ123 pretreatment (p < .01). An inhibition of PAP increase was also observed after diclofenac pretreatment (11.6 +/- 0.4 mm Hg at 5 mins; p < .05). Inhibitory effects at 5 mins were significantly more pronounced in the BQ123 group compared with the diclofenac group. CONCLUSIONS: The effects of ACh on the pulmonary circulation of isolated rabbit lungs depend on ACh concentration and the basal tone of the arterial vasculature. In lungs with a normal pulmonary vascular resistance, ACh administration causes vasoconstriction via the release of ET-1 and TXA2, whereas vasodilation is induced in preconstricted pulmonary vessels.

Platelet-derived thromboxane A2 decreases microvascular perfusion after arterial repair.

Previous work suggests that cod liver oil helps to protect the microcirculation from the consequence of thromboembolic events. The possibility that altered synthesis of thromboxane A2 accounts for the protective effects seen with cod liver oil was investigated in the present study. This was done using the combined thromboxane A2 synthetase inhibitor and thromboxane A2-prostaglandin H2 receptor blocker R68070 (Ridogrel). A standardized microvascular injury was inflicted on the right iliac artery of the rat to generate emboli. The downstream cremaster muscle was used to visualize the passage of the ensuing emboli and to assess the effects of this arterial injury on capillary perfusion and arteriole diameters. The number of visible emboli was not changed by either cod liver oil diet or Ridogrel administration. However, capillary perfusion was preserved by using cod liver oil (n = 7) and was significantly increased by using Ridogrel (n = 7) in comparison with untreated controls (n = 7) in which capillary perfusion was decreased because of the emboli. The administration of Ridogrel to cod liver oil-treated animals (n = 7) provided no additive benefit. The percentage change in A-2 vessel diameters in cod liver oil-treated (n = 7) animals was no different from the control group (n = 7). Ridogrel (n = 7), on the other hand, produced a significant increase in A-3 vessel (n = 21) diameters, but its effects were comparatively less in the cod liver oil-treated animals (n = 7). The formation of platelet aggregates (emboli) appears relatively independent of thromboxane A2 in the rat. Ridogrel is very effective in protecting the microcirculation, and these effects appear to be mediated by A-3 vasodilatation, which, therefore, is at least partially thromboxane A2-dependent. The positive effects of cod liver oil may be mediated by a mechanism that reduces thromboxane A2 synthesis, but further studies are necessary.

Comparative efficacy of nonsteroidal anti-inflammatory drugs and anti-thromboxane agents in a rabbit adhesion-prevention model.

A variety of nonsteroidal anti-inflammatory drugs (NSAIDs) has been found to inhibit postsurgical peritoneal adhesion formation in a number of animal models. A rabbit uterine horn adhesion model was used to directly compare several commonly used NSAIDs of different chemical classes in a single animal study to evaluate their ability to prevent adhesion formation. The effect of thromboxane inhibitors on adhesion prevention was also evaluated. Each of the NSAIDs tested (tolmetin, ibuprofen, aspirin, and indomethacin) showed significant and comparable efficacy. In this same study, imidazole, a thromboxane synthetase inhibitor, also showed significant efficacy. In a second study, ridogrel, an inhibitor of thromboxane synthetase as well as a thromboxane A2 receptor blocker, also showed significant efficacy in reducing peritoneal adhesion severity. These results further support the view that NSAIDs act to prevent adhesions through a common mechanism. In addition, thromboxane A2 inhibitors were also shown to be efficacious in adhesion prevention, suggesting that platelets may play a substantial role in adhesion formation.

Influence of omega-3 fatty acids on splanchnic blood flow and lactate metabolism in an endotoxemic rat model.

Alteration in regional blood flow is important in the pathogenesis of organ failure during endotoxemia and sepsis. In particular, intestinal ischemia is thought to enhance the translocation of bacteria into the systemic circulation. We used radioactive microspheres to measure the influence of two intravenous (IV) dietary fats (vegetable oil containing high levels of omega-6 fatty acids, and fish oil containing high levels of omega-3 fatty acids) on regional blood flow during low-dose Escherichia coli endotoxin infusion (0.1 mg/100 g body weight [BW]) in a rat model. Despite absence of changes in the cardiac output, blood flow rates to the small and large intestines, stomach, and pancreas, and also to the skin and skeletal muscle were significantly reduced after 18 hours of endotoxin infusion in the rats fed standard vegetable oil. Short-term IV feeding during a period of 40 hours with an isonitrogenous, isocaloric nutrient solution containing fish oil as the only lipid source normalized intestinal perfusion and increased blood flow to the liver and spleen. Low-dose endotoxin infusion also resulted in significant increases in glucose, lactate, and pyruvate concentrations. In comparison to standard vegetable fat emulsion, fish oil significantly reduced these parameters. A second experiment was conducted to measure lactate kinetics. Based on the dilution of U-14C-lactate, fish oil feeding was associated with higher lactate clearance than standard vegetable oil feeding during the endotoxin infusion. We conclude that short-term IV feeding with fish oil improves intestinal perfusion and portal blood flow, improves glucose tolerance, and increases lactate clearance in a low-dose endotoxin rat model.

[Effect of OKY-046 on airway hyperresponsiveness induced by ozone in dogs].

To elucidate the possible involvement of thromboxane A2 (TxA2) in airway hyperresponsiveness, we examined the effect of OKY-046, a potent and selective inhibitor of TxA2 synthetase, on airway hyperresponsiveness induced by ozone exposure in dogs. Ozone exposure (3 ppm, 2 hr) markedly increased airway responsiveness to inhaled methacholine without affecting basal respiratory resistance. Although ozone also caused a slight but significant increase in neutrophil number in the bronchoalveolar lavage fluid, there was no correlation between the level of airway hyperresponsiveness and increased neutrophil number. Although OKY-046 significantly inhibited the increases in airway hyperresponsiveness in a dose-dependent manner at doses ranging from 100 to 300 mg/kg, p.o., the compound did not affect the basal airway responsiveness and respiratory resistance at 300 mg/kg, p.o. Inhalation of the subthreshold concentration (i.e., the highest dose which did not cause bronchoconstriction) of STA2 (a stable TxA2 mimetic agent) elicited a significant increase in airway responsiveness to methacholine. These results suggest that TxA2 may play a role in mediating ozone-induced airway hyperresponsiveness. However, the accumulation of neutrophils in the airway lumen may not be essential for the development of airway hyperresponsiveness.

Role of thromboxane A2 in the control of myocardial O2 supply/consumption balance and severity of ischemia during pacing-induced ischemia.

The role of thromboxane A2 (TXA2) in the control of O2 supply/consumption variables during pacing-induced ischemia was examined using the TXA2 receptor antagonist SQ 29,548. Anesthetized, open-chest dogs were subjected to left anterior descending coronary artery (LAD) stenosis that produced significant epicardial S-T segment elevation (12 mV) only when superimposed on atrial pacing. Regional myocardial blood flow was determined using radioactive microspheres, and O2 consumption was determined by measuring O2 saturation of venous blood draining the ischemic region. The dogs were treated with saline or 0.2 mg/kg + 0.2 mg/kg/hr SQ 29,548, and the effect on ischemia was determined during 5-minute pacing-induced ischemic episodes at 10, 40, and 70 minutes postdrug or saline treatment. SQ 29,548 significantly reduced S-T elevation at 40 and 70 minutes postdrug compared with saline values and at all times measured compared with its paired predrug pace+stenosis values. SQ 29,548 reduced S-T elevation approximately 45% compared with its paired predrug values at 70 minutes. SQ 29,548 resulted in a significantly higher subendocardial-to-subepicardial flow ratio (0.70 +/- 0.10, p less than 0.05) compared with saline-treated animals (0.42 +/- 0.06), with an overall increase of flow to the ischemic region of approximately 40%. This increased flow was matched by a proportional increase in O2 consumption without a change in O2 extraction. The O2 supply/consumption balance was also unchanged by SQ 29,548 implying that despite the increase in blood flow, the ischemic region was still flow-limited.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 and fever: a continuing debate.

Prostaglandin (PG) E2 is a potent hyperthermic agent and has been assigned an intermediary function in the response of thermoregulatory neurons to pyrogens. Though attractive, this idea has been challenged on several grounds. The present study confirms that brain PGE2 synthesis increases during fever, the time course of the elevation according with a causative role of the compound. Our experimental data also argue against the involvement of a second cyclooxygenase product, specifically thromboxane (TX) A2, in the action of pyrogens. The sequence of events leading to PGE2 production and fever differs depending on the pyrogen, bacterial vs. leucocytic, and its route of administration. Blood-borne interleukin-1 (IL-1) acts on a discrete site in the central nervous system (CNS) which is tentatively identified with the organum vasculosum laminae terminalis (OVLT). The same site may also be the target for blood-borne endotoxin. In addition, endotoxin may promote PGE2 synthesis in the cerebral microvasculature. Both pyrogens, on the other hand, act diffusely throughout the CNS when given intrathecally. We conclude that PGE2 is well suited for an intermediary role in the genesis of fever and ascribe the reported inconsistencies to methodological factors.

Multiple sclerosis: the rational basis for treatment with colchicine and evening primrose oil.

Multiple sclerosis (MS) is a disease with no known treatment. In view of this and of its distressing nature patients are attracted by any new concepts. As a reaction to this neurologists are sometimes excessively sceptical and fail to consider new approaches seriously. Recent attempts have been made to treat multiple sclerosis with polyunsaturated fatty acids and with colchicine. This approach is not arbitrary and is firmly grounded in fundamental basic scientific concepts. In patients with multiple sclerosis there is evidence of both an abnormality in essential fatty acid metabolism and an abnormality in lymphocyte function. It is now apparent that the fatty acid abnormality may cause the lymphocyte abnormality and that both may be improved by dietary manipulation. There is also evidence that the demyelination may be associated with recurrent inflammatory episodes and with entry of calcium into the cytoplasm. In vitro colchicine has been shown to have actions compatible with regulation of cytoplasmic calcium and in two diseases characterised by intermittent inflammatory episodes (Behçets syndrome and familial Mediterranean fever) it has been found to prevent or to reduce the severity of such episodes. Preliminary results suggest that combined therapy with evening primrose oil and colchicine may be of considerable value.