Thromboxane A2 Synthase Inhibitor Research Articles

Antiplatelet activity of J78 (2-Chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), an antithrombotic agent, is mediated by thromboxane (TX) A2 receptor blockade with TXA2 synthase inhibition and suppression of cytosolic Ca2+ mobilization.

We previously reported that J78 (2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, exhibited a potent antithrombotic effect, which might be due to antiplatelet rather than anticoagulation activity. In the present study, possible anti-platelet mechanism of J78 was investigated. J78 concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/ml), thrombin (0.05 U/ml), arachidonic acid (100 microM), and U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F(2); 1 microM), a thromboxane (TX) A(2) mimic, with IC(50) values of 0.32 +/- 0.01, 0.44 +/- 0.02, 0.50 +/- 0.04, and 0.36 +/- 0.02 microM, respectively. J78 also produced a shift to the right of the concentration-response curve of U46619, indicating an antagonistic effect on the TXA(2) receptor. J78 concentration-dependently inhibited collagen-induced arachidonic acid liberation. In addition, J78 potently suppressed TXA(2) formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner but had no effect on the production of PGD(2), indicating an inhibitory effect on TXA(2) synthase. This was supported by a TXA(2) synthase activity assay that J78 concentration-dependently inhibited TXB(2) formation converted from PGH(2). Furthermore, J78 was also able to inhibit the [Ca(2+)](i) mobilization induced by collagen or thrombin at such a concentration that completely inhibited platelet aggregation. Taken together, these results suggest that the antiplatelet activity of J78 may be mediated by TXA(2) receptor blockade with TXA(2) synthase inhibition and suppression of cytosolic Ca(2+) mobilization.

Functional role of NADPH oxidase in activation of platelets.

Involvement of phagocyte NADPH oxidase in host defense response is well established. In contrast, little is known about the functional role of NADPH oxidase in platelets. In this study, we analyzed involvement of platelet NADPH oxidase in aggregation of human platelets and in amplification of production of reactive oxygen species (ROS) by activated human neutrophils. Apocynin, a known NADPH oxidase inhibitor, as well as superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-1-pyridyl)porphyrin, inhibited ROS generation by collagen-activated platelets, collagen-induced aggregation of platelets, as well as collagen-induced release of thromboxane B2. These data suggest the key role of intracellular ROS derived from NADPH oxidase in the control of thromboxane A2 (TXA2) production in platelets stimulated by collagen. Apocynin also inhibited thrombin-induced ROS production and thrombin-induced platelet aggregation. Activation of neutrophils with latex resulted in an outburst of ROS that was inhibited by apocynin. ROS production by latex-stimulated platelets was modest and also inhibited by apocynin. However, when a mixture of platelets and neutrophils was stimulated with latex, ROS production was three to six times higher in comparison with activation of neutrophils alone. Platelet-dependent augmentation of neutrophil ROS production was abrogated by TXA2 synthase inhibitor (furegrelate, 1 microM) or by aspirin (300 microM). In summary, NADPH oxidase in platelets seems to play a major role as an intracellular signaling mechanism in the activation of platelets. However, in host defense response involving neutrophils and platelets, platelets enhance ROS production by neutrophils and possibly their cytotoxic potential via the release of TXA2, which in turn in platelets is not affected by the extracellular release of free radicals.

Ramatroban (BAY u 3405): a novel dual antagonist of TXA2 receptor and CRTh2, a newly identified prostaglandin D2 receptor.

It is known that thromboxane A2 (TXA2) contributes to various diseases such as bronchial asthma, ischemic heart disease, cerebrovascular disorders and allergic rhinitis. A number of TXA2 synthase inhibitors and TXA2 receptor (TP receptor) antagonists have been developed to treat these diseases. Ramatroban (BAY u 3405) was developed as a potent TP receptor antagonist with excellent efficacy against allergic rhinitis in many animal models and patients. Recent studies also revealed that ramatroban can block the newly identified PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2). PGD2 induces migration and degranulation of eosinophils through CRTh2 and contributes to late-phase inflammation and cell damage. Accordingly, it was considered that ramatroban suppresses the late-phase inflammation via TP receptor and CRTh2 blockade. In terms of the efficacy on vascular systems, it was revealed that ramatroban can suppress the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in endothelial cells and prevent exacerbation of inflammation by blocking these responses. According to our recent studies in hypercholesterolemic rabbits ramatroban prevents macrophage infiltration through MCP-1 downregulation and neointimal formation after balloon injury and attenuates vascular response to acetylcholine. Therefore, ramatroban may be beneficial in the treatment of atherosclerosis.

Cytotoxicity of the E2-isoprostane 15-E2t-IsoP on oligodendrocyte progenitors

Oxidant stress plays a significant role in the pathogenesis of periventricular leukomalacia (PVL). Isoprostanes (IsoPs) are bioactive products of lipid peroxidation abundantly generated during hypoxic–ischemic injuries. Because loss of oligodendrocytes (OLs) occurs early in PVL, we hypothesized that IsoPs could induce progenitor OL death. 15-E2t-IsoP but not 15-F2t-IsoP elicited a concentration-dependent death of progenitor OLs by oncosis and not by apoptosis, but exerted minimal effects on mature OLs. 15-E2t-IsoP-induced cytotoxicity could not be explained by its conversion into cyclopentenones, because PGA2 was hardly cytotoxic. On the other hand, thromboxane A2 (TxA2) synthase inhibitor CGS12970 and cyclooxygenase inhibitor ibuprofen attenuated 15-E2t-IsoP-induced cytotoxicity. Susceptibility of progenitor OLs was independent of TxA2 receptor (TP) expression, which was far less in progenitor than in mature OLs. However, TxA2 synthase was detected in precursor but not in mature OLs, and TxA2 mimetic U46619 induced hydroperoxides generation and progenitor OL death. The glutathione synthesis enhancer N-acetylcysteine prevented 15-E2t-IsoP-induced progenitor cell death. Depletion of glutathione in mature OLs with buthionine sulfoximine rendered them susceptible to cytotoxicity of 15-E2t-IsoP. These novel data implicate 15-E2t-IsoP as a product of oxidative stress that may contribute in the genesis of PVL.

Involvement of thromboxane A2 (TXA2) in the early stages of oleic acid-induced lung injury and the preventive effect of ozagrel, a TXA2 synthase inhibitor, in guinea-pigs.

An intravenous injection of oleic acid into animals can produce a lung injury with hypoxaemia and pulmonary vascular hyper-permeability. Although oleic acid lung injury is used as a model of acute respiratory distress syndrome (ARDS), the precise mechanisms of the lung injury are still unclear. We have investigated whether thromboxane A(2) (TXA(2)) participated in the lung injury and have evaluated the efficacy of ozagrel, a TXA(2) synthase inhibitor, on the lung injury in guinea-pigs. Oleic acid injection increased the plasma level of TXB(2), a stable metabolite of TXA(2), and the time-course of plasma TXB(2) was similar to that of the decreased partial oxygen pressure of arterial blood (Pao(2)) induced with oleic acid. Ozagrel administered intravenously 30 min before oleic acid injection prevented the decrease in Pao(2) and pulmonary vascular hyper-permeability. It also prevented increases in lactate dehydrogenase activity, a measure of lung cell injury, TXB(2 )and its weight ratio to 6-keto prostaglandin F(1alpha) in bronchoalveolar lavage fluid. Although ozagrel administered simultaneously with oleic acid ameliorated the decrease in Pao(2), post treatment showed little effect. We suggest that TXA(2) participated in the oleic acid lung injury, as an "early phase" mediator, and rapidly-acting TXA(2) synthase inhibitors were effective in the prevention of acute lung injury.

Effect of a novel thromboxane A2 inhibitor on right ventricular-arterial coupling in endotoxic shock.

We investigated the effects of a dual thromboxane (TX)A2 synthase inhibitor and TXA2 receptor antagonist (BM-573) on right ventricular-arterial coupling in a porcine model of endotoxic shock. Thirty minutes before the onset of 0.5 mg/kg endotoxin infusion, six pigs (Endo group) received an infusion with a placebo solution, and six other pigs (Anta group) with BM-573. Right ventricular pressure-volume loops were obtained by the conductance catheter technique. The slope (Ees) of the end-systolic pressure-volume relationship and its volume intercept at 25 mmHg were calculated as measures of right ventricular systolic function. RV afterload was quantified by pulmonary arterial elastance (Ea), and Ees/Ea ratio represented right ventricular-arterial coupling. Mechanical efficiency was defined as the ratio of stroke work and pressure-volume area. In this model of endotoxic shock, BM-573 blunted the early phase of pulmonary hypertension, improved arterial oxygenation, and prevented a decrease in right ventricular myocardial efficiency and right ventricular dilatation. However, the drug could not prevent the loss of homeometric regulation and alterations in right ventricular-arterial coupling. In conclusion, dual TXA2 synthase inhibitor and receptor antagonists such as BM-573 have potential therapeutic applications, improving right ventricular efficiency and arterial oxygenation in endotoxic shock.

Mechanisms involved in the early increase of serotonin contraction evoked by endotoxin in rat middle cerebral arteries.

The present study investigated the mechanisms involved in the increased 5-hydroxytryptamine (5-HT) vasoconstriction observed in rat middle cerebral arteries exposed in vitro to lipopolysaccharide (LPS, 10 microg x ml-1) for 1-5 h. Functional, immunohistochemical and Western blot analysis and superoxide anion measurements by ethidium fluorescence were performed. LPS exposure increased 5-HT (10 microm) vasoconstriction only during the first 4 h. In contrast to control tissue, indomethacin (10 microm), the COX-2 inhibitor NS 398 (10 microm), the TXA2/PGH2 receptor antagonist SQ 29548 (1 microm) and the TXA2 synthase inhibitor furegrelate (1 microm) reduced 5-HT contraction of LPS-treated arteries from hour one. The iNOS inhibitor aminoguanidine (0.1 mm) increased 5-HT contraction from hour three of LPS incubation. The superoxide anion scavenger superoxide dismutase (SOD, 100 U ml-1) and the H2O2 scavenger catalase (1000 U ml-1), as well as the respective inhibitors of NAD(P)H oxidase and xanthine oxidase, apocynin (0.3 mm) and allopurinol (0.3 mm), reduced 5-HT contraction after LPS incubation. LPS induced an increase in superoxide anion levels that was abolished by PEG-SOD. Subthreshold concentrations of the TXA2 analogue U 46619, xanthine/xanthine oxidase and H2O2 potentiated, whereas those of sodium nitroprusside inhibited, the 5-HT contraction. COX-2 expression was increased at 1 and 5 h of LPS incubation, while that of iNOS, Cu/Zn-SOD and Mn-SOD was only increased after 5 h. All the three vascular layers expressed COX-2 and Cu/Zn-SOD. iNOS expression was detected in the endothelium and adventitia after LPS. In conclusion, increased production of TXA2 from COX-2, superoxide anion and H2O2 enhanced vasoconstriction to 5-HT during the first few hours of LPS exposure; iNOS and SOD expression counteracted that increase at 5 h. These changes can contribute to the disturbance of cerebral blood flow in endotoxic shock.

Obligatory role of lipid mediators in platelet–neutrophil adhesion

Platelet–neutrophil interactions play an important role in thrombotic and inflammatory responses. Although it is well known that adhesion of platelets to neutrophils requires interactions of adhesion molecules on platelets such as P-selectin, or GPIIb/IIIa with their counterparts on neutrophils, little is known on the role of lipid mediators in this response. Here we studied involvement of thromboxane (TX) A2, platelet activating factor (PAF) and cysteinyl leukotrienes (cysLTs) in the mechanisms of platelet–neutrophil adhesion that was induced by thrombin (10–100 mU/ml), fMLP (0.01–1 μM) or LPS (0.001–100 μg/ml). All three stimulators in a concentration- and time-dependent manner induced platelet–neutrophil adhesion as quantified by the method of Jungi et al. [Blood 67(3) (1986) 629]. Platelet–neutrophil adhesion induced by each of the three activators was inhibited by blocking antibodies towards P-selectin, GPIIb/IIIa or CD18, but it was not affected by anti-E selectin antibody. Moreover, platelet–neutrophil adhesion induced by thrombin, fMPL or LPS was inhibited by the inhibitor of cyclooxygenase (aspirin), by TXA2 synthase inhibitor (camonagrel), by PAF receptor antagonist (WEB 2170), by the inhibitor of FLAP (MK 886) and by cysLTs receptors antagonist (MK 571). On the other hand, the selective inhibitor of COX-2 (rofecoxib) as well as the inhibitor of cytochrome P450-dependent monoxygenase (17-ODYA) were ineffective.In summary, adhesion of platelets to neutrophils is regulated not only by specific interaction between adhesion molecules on platelets and neutrophils, but also by lipid mediators such as TXA2, PAF and cysLTs released upon activation of platelets or/and neutrophils.

Increased platelet-activating factor-induced periventricular brain microvascular constriction associated with immaturity.

Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on periventricular (PV) microvessels of fetal ( approximately 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 +/- 2.6%) and newborn (22.14 +/- 3.2%) pigs but was ineffective in adults (<2.5%). Specific [(3)H]PAF binding was greater in fetus and newborn than in adults; a concordant developmental PAF-induced inositol phosphate formation was observed. PAF-induced vasoconstriction was abrogated by thromboxane A(2) (TXA(2)) synthase and receptor inhibitors, calcium channel blockers, and by removal of endothelium; vasoconstriction to TXA(2) mimetic U-46619 did not differ with age. Immunoreactive TXA(2) synthase expression and PAF-evoked TXA(2) formation revealed a fetus> newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TXA(2) formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects.

Selective neuromicrovascular endothelial cell death by 8-Iso-prostaglandin F2alpha: possible role in ischemic brain injury.

Free radical-induced peroxidation is an important factor in the genesis of hypoxic-ischemic encephalopathy, including that of the preterm infant. Isoprostanes are major peroxidation products. Since microvascular dysfunction seems to contribute to ischemic encephalopathies, we studied the cytotoxicity of 8-iso-prostaglandin F2alpha (PGF2alpha) on cerebral microvascular cells. Microvascular endothelial, astroglial, and smooth muscle cells from newborn brain were cultured. The cytotoxicity of 8-iso-PGF2alpha on these cells was determined by MTT assays and lactate dehydrogenase (LDH) release, propidium iodide incorporation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]). In addition, effects of intraventricular injections of 8-iso-PGF2alpha and possible involvement of thromboxane in 8-iso-PGF2alpha-induced cytotoxicity were determined. 8-Iso-PGF2alpha induced time- and concentration-dependent endothelial cell death (EC50=0.1 nmol/L) but exerted little effect on smooth muscle and astroglial cells; endothelial cell death seemed mostly of oncotic nature (propidium iodide incorporation and LDH release). Cell death was associated with increased endothelial thromboxane A2 (TXA2) formation and was prevented by TXA2 synthase inhibitors (CGS12970 and U63557A); TXA2 mimetics U46619 and I-BOP also caused endothelial cell death. Intraventricular injection of 8-iso-PGF2alpha induced periventricular damage, which was attenuated by CGS12970 pretreatment. These data disclose a novel action of 8-iso-PGF2alpha involving TXA2 in oxidant stress-induced cerebral microvascular injury and brain damage.

20-HETE-induced contraction of small coronary arteries depends on the activation of Rho-kinase.

20-HETE is a potent constrictor of small blood vessels and has been suggested to play a crucial role in the generation of myogenic tone and the development of hypertension. In the present study, we investigated the mechanisms by which exogenously applied 20-HETE modulates vascular tone in small porcine coronary arteries. In organ chamber experiments, 20-HETE elicited a concentration-dependent contraction of small porcine coronary artery rings that was partially inhibited by the cyclooxygenase inhibitor diclofenac, the thromboxane and endoperoxide receptor antagonist SQ29548, and the thromboxane A2 synthase inhibitor furegrelate. Removal of endothelium attenuated the response to 20-HETE, whereas preconstriction of endothelium-denuded vessels to 25% of the maximum response with KCl markedly enhanced the response to 20-HETE. This 20-HETE-induced contraction was not associated with a significant increase in the intracellular concentration of Ca2+. 20-HETE-induced contraction was also observed in beta-escin-permeabilized arteries precontracted with a submaximal concentration of Ca2+ and was abolished by the Rho-kinase inhibitor Y27632, but was insensitive to the PKC inhibitor RO 31-8220. 20-HETE elicited the phosphorylation of the myosin light chain (MLC20) in coronary artery rings, an effect that was sensitive to Y27632 and mimicked by the thromboxane analog U46619. These data suggest that in small porcine coronary arteries, 20-HETE can induce contraction by 2 mechanisms, one endothelium-dependent involving the cyclooxygenase-dependent generation of vasoconstrictor prostanoids, and the other endothelium-independent. The latter response is associated with the activation of Rho-kinase, phosphorylation of MLC20, and sensitization of the contractile apparatus to Ca2+.

Downregulation of nitric oxide accumulation by cyclooxygenase-2 induction and thromboxane A2 production in interleukin-1beta-stimulated rat aortic smooth muscle cells.

Cytokines from inflammatory cells do not produce nitric oxide, but stimulate the production of nitric oxide in vascular smooth muscle cells (VSMC). Thromboxane A2 (TXA2) has been believed to have a key role in atherosclerogenesis and post-angioplasty restenosis. To determine whether cytokine-induced nitric oxide production is regulated by the TXA2/prostaglandin H2 (PGH2) receptor. We studied the interleukin-1beta (IL-1beta)-induced production of nitric oxide in rat VSMCs using the TXA2/PGH2 receptor antagonists, seratrodast and Bay-u3405, and an agonist, U-46619. Nitrite formation was measured colorimetrically. IL-1beta increased nitrite formation in a time-dependent manner. The nitrite concentration was 1.7 times greater in the presence of seratrodast than that without it. Nitrite accumulation was increased by Bay-u3405, but was decreased in the presence of U-46619, to 44% of that in its absence. Western and Northern blotting showed that seratrodast increased the levels of expression of inducible nitric oxide synthase (iNOS) protein and mRNA in a dose-dependent manner, whereas U-46619 decreased them. We speculated that VSMCs produced TXA2, thereby decreasing nitric oxide production; therefore we measured the accumulation of TXB2 using an enzyme immunoassay. Untreated VSMCs produced about 20 pg/mg protein of TXB2. This was increased by the addition of IL-1beta, to 152.1 +/- 43.0 pg/mg protein after a 24 h incubation; the expression of cyclooxygenase-2 (COX-2) protein was also increased, but there was no effect on the expression of COX-1 and TXA2 synthase. U-63557A, a TXA2 synthase inhibitor, increased the accumulation of nitrite to 1.3-fold that in its absence. These data suggest that the expression of iNOS and the production of nitric oxide are regulated by the TXA2/PGH2 receptor in IL-1beta-stimulated VSMCs. The endogenous production of TXA2 by the induction of COX-2 from IL-1beta-stimulated VSMCs probably downregulated the production of nitric oxide in VSMCs. TXA2/PGH2 receptor inhibitors may contribute to the reduction in formation of atherosclerosis in lesions with vascular injury by enhancing the production of nitric oxide by VSMCs.

2,3-Dinor-5,6-dihydro-15-F(2t)-isoprostane: a bioactive prostanoid metabolite.

15-F(2t)-isoprostane (15-F(2t)-IsoP), also termed 8-isoprostaglandin F(2alpha), is one of a series of prostanoids formed by free radical-mediated peroxidation of arachidonic acid and exerts potent biological actions such as vasoconstriction. We recently demonstrated that 15-F(2t)-IsoP is metabolized in humans to a major metabolite, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (15-F(2t)-IsoP-M). 15-F(2t)-IsoP-M can also potentially be formed as a product of free radical-induced oxidation of the low abundance fatty acid gamma-linolenic acid. We confirmed that 15-F(2t)-IsoP-M is generated during oxidation of gamma-linolenic acid and explored whether it may exhibit biological activity. 15-F(2t)-IsoP-M caused marked constriction of porcine surface retinal and intraparenchymal brain microvessels, comparable to that observed with 15-F(2t)-IsoP. These effects were associated with increased thromboxane A(2) (TXA(2)) formation and were virtually abolished by TXA(2)-synthase and -receptor inhibitors (CGS-12970 and L-670596). Vasoconstriction induced by either 15-F(2t)-IsoP or 15-F(2t)-IsoP-M on perfused ocular choroid was also abrogated by TXA(2)-synthase inhibition as well as by removal of endothelium. Similar to 15-F(2t)-IsoP, 15-F(2t)-IsoP-M evoked vasoconstriction and TXA(2) generation by activating Ca(2+) influx from nonvoltage-gated channels (SK&F96365 sensitive) in the retina and from both nonvoltage- and N-type voltage-gated Ca(2+) channels (omega-conotoxin MVIIA sensitive), respectively, in brain endothelial and astroglial cells; smooth muscle cells were unresponsive to both agents. Cross-desensitization experiments further suggest that 15-F(2t)-IsoP and 15-F(2t)-IsoP-M act on the same receptor mechanism. Findings reveal a novel concept by which a beta-oxidation metabolite of 15-F(2t)-IsoP that can also be formed by nonenzymatic oxidation of gamma-linolenic acid is equivalently bioactive to 15-F(2t)-IsoP and may prolong the vascular actions of F(2)-IsoPs.

Thrombin inhibition attenuates neurodegeneration and cerebral edema formation following transient forebrain ischemia

The disturbance of microcirculation following cerebral ischemia leads to an enlargement of cerebral infarct volume. Endogenous thrombin may play a role in this disturbance of microcirculation following cerebral ischemia. Therefore, the inhibition of thrombin may improve neurodegeneration and the accumulation of cerebral edema following cerebral ischemia in gerbils. The effects of thrombin inhibitor (argatroban) on cerebral ischemia were investigated in comparison with thromboxane A2 synthase inhibitor (ozagrel) and cyclooxygenase inhibitor (aspirin) following bilateral common carotid artery occlusion and reperfusion (CCA:O/R) in male Mongolian gerbils. This study consisted of three experiments: (1) morbidity and survival ratio ( n=40 for each), (2) histopathology ( n=12 for each), and (3) mean arterial blood pressure, local cerebral blood flow (CBF), and cerebral specific gravity ( n=8 for each). Argatroban treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex and hippocampus and cerebral edema in cortex compared with aspirin and saline, in concert with the fast recovery of local CBF without reactive hyperemia following bilateral CCA:O/R. Ozagrel treatment also improved those factors compared with saline, in concert with the fast recovery of local CBF with reactive hyperemia. Aspirin treatment improved survival ratio and stroke index, and decreased ischemically injured cell numbers in cortex. Thrombin inhibition with argatroban decreases neurodegeneration and cerebral edema following bilateral CCA:O/R in gerbils.

Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy.

Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A2 (TxA2), we tested whether TxA2 plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O2 exposure from postnatal days 5-14) was associated with increased TxB2 generation and was significantly prevented by TxA2 synthase inhibitor CGS-12970 (10 mg x kg(-1) x day(-1)) or TxA2-receptor antagonist CGS-22652 (10 mg x kg(-1) x day(-1)). TxA2 mimetics U-46619 (EC50 50 nM) and I-BOP (EC50 5 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF2, which is generated in OIR, stimulated TxA2 formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA2-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA2 in vasoobliteration of OIR and unveil a so far unknown function for TxA2 in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA2 might participate in other ischemic neurovascular injuries.

Role of prostanoids and endothelins in the prevention of cyclosporine-induced nephrotoxicity

Cyclosporine A nephrotoxicity includes both functional toxicity and histological changes, whose seriousness is dependent upon the dose and the duration of the drug administration. Several vasoactive agents have been found to be implicated in cyclosporine induced nephrotoxicity, among which prostanoids and endothelins are the most important. In previous studies we were able to prevent the early stage (7 days) of cyclosporine (37.4 μmol [45 mg]/kg/day) induced nephrotoxicity in rats either by the administration, i) of OKY-046, a thromboxane A2synthase inhibitor, ii) of ketanserine, an antagonist of S2serotonergic, a1adrenergic, and H1histaminergic receptors and iii) of nifedipine, a calcium channel blocker, or by diet supplementation either with evening primrose oil or fish oil. All these protective agents elevated ratios of excreted renal prostanoid vasodilators (prostaglandins E2, 6ketoF1α) to vasoconstrictor (thromboxane B2), a ratio which was decreased by the administration of cyclosporine alone. Nifedipine averted the cyclosporine induced increase of urinary endothelin-1 release. All protections were associated with the reinstatement of glomerular filtration rate forwards normal levels whereas renal damage defence, consisting of a decrease of the cyclosporine induced vacuolizations, was variable. Ketanserine and evening primrose oil were the only agents which prevented the animal body weight loss. These data suggest that prostanoids and endothelin-1 may mediate functional toxicity while thromboxane A2is involved the morphological changes too, provoked in the early stage of cyclosporine treatment. However, other nephrotoxic factors and additional mechanisms could also be implicated in the cyclosporine induced nephrotoxicity.

Flow-induced constriction in arterioles of hyperhomocysteinemic rats is due to impaired nitric oxide and enhanced thromboxane A(2) mediation.

Hyperhomocysteinemia (HHcy) is thought to promote arteriosclerosis and peripheral arterial disease, in part by impairing the function of endothelium. Because flow-induced dilation is mediated by the endothelium, we hypothesized that HHcy alters this response by interfering with the synthesis/action of NO and prostaglandins. Thus, changes in the diameter of isolated, pressurized (at 80 mm Hg) gracilis skeletal muscle arterioles (diameter approximately 170 microm) from control and methionine diet-induced HHcy rats were investigated with videomicroscopy. Increases in intraluminal flow (from 0 to 25 microL/min) resulted in dilations of control arterioles (maximum, 34+/-4 microm). In contrast, increases in flow elicited constrictions of HHcy arterioles (-36+/-3 microm). In control arterioles, the NO synthase inhibitor N:(omega)-nitro-L-arginine-methyl ester significantly attenuated (approximately 50%) dilation, whereas the additional administration of indomethacin, an inhibitor of cyclooxygenase, eliminated flow-induced dilation. In the arterioles of HHcy rats, flow-induced constriction was not affected by N:(omega)-nitro-L-arginine-methyl ester, whereas it was abolished by indomethacin or the prostaglandin H(2)/thromboxane A(2) (TXA(2)) receptor antagonist SQ 29,548 or the TXA(2) synthase inhibitor CGS 13,080. Thus, in HHcy, increases in intraluminal flow elicit constrictions of skeletal muscle arterioles due to the impaired NO and enhanced TXA(2) mediation of the response, alterations that likely contribute to the development of peripheral arterial disease.

Serotonin-induced human coronary microvascular contraction during acute myocardial ischemia is blocked by COX-2 inhibition.

Since serotonin (5-HT) is implicated in exacerbating acute coronary syndromes, we studied the reactivity of atrial coronary arterioles (70-140 microm) of atherosclerotic patients undergoing cardiac surgery to 5-HT, substance P (Sub P), and sodium nitroprusside by video-microscopy. Before ischemia, 5-HT-induced relaxation was not affected by NS398 (cyclooxygenase inhibitor), H2O2 or U63557A (thromboxane A2 synthase inhibitor), but was reduced by L-NNA. 5-HT elicited a potent contractile response after ischemia that was inhibited by NS398, Indo, and U63557A. While Sub P relaxation was decreased after ischemia, SNP relaxation was unchanged. The mRNA steady-state levels of NOS-3, NOS-2, prostacyclin synthase, and COX- 1 were not altered by ischemia. COX-2 mRNA and protein levels (Westernblotting), however, were increased (mean +/- SEM) 2.4 +/- 0.4 and 3.2 +/- 0.7 fold, respectively, in ischemic atrium corroborating with the immunohistochemistry of atrial tissue. It is concluded that myocardial ischemia enhanced contractile response of coronary arterioles to 5-HT maybe due to the stimulated prostaglandin release (likely thromboxane A2) secondary to induction of COX-2 expression. These findings may have implications regarding the cause of coronary spasm during acute myocardial ischemia.

NQ-Y15 inhibits the calcium mobilization by elevation of cyclic AMP in rat platelets.

2-1(4-Cyanophenyl)aminol-3-chloro-1,4-naphthalenedione (NQ-Y15) is a dual action drug which acts as a thromboxane A2 (TXA2) synthase inhibitor and TXA2/PGH2 receptor antagonist. In the present study, we examined the effects of NQ-Y15 on Ca2+ mobilization, which is the common event in various types of platelet activation, in arachidonic acid (AA)-stimulated rat platelets. The elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by AA was inhibited by NQ-Y15 in a concentration-dependent manner. This inhibition-effect of NQ-Y15 was found to be based on the suppression of the rise in [Ca2+]i by the inhibition of both Ca2+ release from internal stores and Ca2+ influx from the extracellular space. Our successive trial was focused on the role of cyclic AMP (cAMP) in the action of NQ-Y15, because cAMP was reported to be increased by dual action drugs such as picotamide and to inhibit the increase in [Ca2+]i. NQ-Y15 was confirmed to increase cAMP in AA-stimulated rat platelets. These results suggested that NQ-Y15 might inhibit the rise in [Ca2+]i in AA-treated rat platelets by increasing cAMP, which is involved in the inhibition of platelet activation.

Multiple endothelial injury in epicardial coronary artery induces downstream microvascular spasm as well as remodeling partly via thromboxane A2

OBJECTIVESThe study was undertaken to develop a coronary microvascular spasm model in pigs by repeated epicardial coronary artery endothelial injury.BACKGROUNDThe pathophysiologic mechanisms responsible for coronary microvascular spasm remain unclear, in large part because a suitable animal model has yet to be found.METHODSBalloon endothelial denudation was done just distal to the site of an implanted Doppler flowmeter in the left anterior descending coronary artery (LAD) every two weeks for a total of four times. Changes in LAD blood flow by intracoronary administration of vasoactive agents were assessed before each denudation.RESULTSIn the epicardial LAD endothelial denudation pigs, decreases in LAD blood flow caused by acetylcholine were augmented. Before denudation, it was −15 ± 4%, and at week 8 (i.e., two weeks after the fourth denudation) it was −100% (i.e., zero flow [p < 0.01]). The LAD flow changes in response to 5-hydroxytryptamine (5-HT) changed from an increase to a decrease, accompanied by medial thickening of microvessels in the LAD perfusion area. These flow responses were observed without significant changes in LAD diameter. In contrast, the LAD blood flow responses to acetylcholine and 5-HT did not change throughout the experiment in pigs given aspirin and a thromboxane A2(TXA2) synthase inhibitor orally.CONCLUSIONSThis microvascular spasm model indicates that hypersensitivity to vasoactive substances in the microvascular beds as well as microvascular remodeling are brought about partly through TXA2. This model should be useful for examining the pathophysiology and treatment of microvascular angina.