Thromboxane Metabolites Research Articles

Parecoxib, Valdecoxib, and Cardiovascular Risk

Selective inhibitors of cyclooxygenase (COX)-21 depress prostacyclin (PGI2) but not COX-1–derived thromboxane A2. The effects of thromboxane A2 would be exaggerated during treatment with COX-2 inhibitors, potentially predisposing patients to heart attack and stroke.2 A randomized controlled trial in patients undergoing coronary artery bypass graft (CABG) surgery and receiving either placebo or 40 mg parecoxib (Dynastat; Pfizer), the intravenously administered prodrug of valdecoxib (Bextra; Pfizer), followed by oral valdecoxib 40 mg BID for 14 days, revealed a cluster of cardiovascular events.3 In a second study, one group received parecoxib/valdecoxib, another …

Prostacyclin, thromboxane A2 and the effect of low‐dose ASA in pregnancies at high risk for hypertensive disorders

The aim of this study was to investigate the prostanoid production in pregnancies at high risk for hypertensive disorders, and the effect of low-dose acetylsalicylic acid (ASA) on prostanoids. Ninety women with a bilateral notching in uterine arteries screened by Doppler ultrasound at 12-14 gestational weeks were randomized to the ASA (0.5 mg/kg/day) or placebo group. Forty-three women in both groups were followed up throughout the pregnancy. Urine samples were taken at baseline, and at 24-26 and 32-34 weeks of gestation to determine the urinary 11-dehydrothromboxane B(2) (u-11-dehydro-TxB(2)) and 2,3-dinor-6-keto-prostaglandin F(1alpha) (u-2,3-dinor-6-keto-PGF(1alpha)), the metabolites of thromboxane A(2) and prostacyclin, respectively. In the pregnancies with pregnancy-induced hypertension (PIH) before 37 gestational weeks, the 2,3-dinor-6-keto-PGF(1alpha)/11-dehydro-TxB(2) ratio did not increase as much as in other pregnancies (P = 0.028). In the placebo group pregnancies with preeclampsia had significantly lower 2,3-dinor-6-keto-PGF(1alpha) (P = 0.019) at 12-14 weeks of gestation compared to other pregnancies. In the placebo group the 2,3-dinor-6-keto-PGF(1alpha)/11-dehydroTxB(2) ratio remained unchanged throughout the pregnancy, with no significant difference between pregnancies with a normal or an adverse outcome. In the ASA group the 2,3-dinor-6-keto-PGF(1alpha)/11-dehydro-TxB(2) ratio increased (P < 0.001, early vs. midpregnancy). Again, the changes were similar in pregnancies with a normal or an adverse outcome. The balance of prostacyclin and thromboxane A(2) shifted in an unfavorable direction in pregnancies complicated by PIH. ASA had a favorable effect on the prostanoids.

Comparison of Three Different Platelet Function Assays to Determine Aspirin Sensitivity.

In large studies of patients with cardiac disease, it was observed that up to 9% are ASA resistant (ASAR) and 23% are ASA semi responders using the platelet function analyzer (PFA-100), which measures whole blood platelet adhesion and aggregation under high shear. The prevalence of ASAR is of great clinical importance. With an estimated 20 million patients in the USA taking ASA for prevention of atherosclerotic events, even a 10% incidence equates to more than two million patients receiving inadequate anti-platelet therapy. Given the widespread use of ASA, more reliable and rapid methods to measure aspirin sensitivity are needed. Importantly, future large scale studies to determine the effect of monitoring ASA sensitivity to optimize therapy are compromised due to current lack of uniformity in assessing ASAR from center to center. The classical method of platelet aggregometry is labor-intensive and not readily adaptable to the clinical setting. Moreover, platelet aggregometry measures platelet responses under low shear, which does not simulate the high shear conditions expected to be involved in arterial thrombosis. In contrast, the PFA-100 does measure thrombus formation under high shear. The new TEG platelet coagulation assay uses whole blood, includes high shear, and is a point of care method. We compared these three techniques to assess aspirin effects on platelet function and clot formation: PFA-100 with collagen-epinephrine cartridges, TEG platelet coagulation assay, and standard optical aggregation in platelet rich plasma using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation. Twelve normal individuals previously defined as ASA sensitive or ASAR, using the PFA-100, were studied at baseline and following three days of oral aspirin at 81mg/day. We found that all four patients determined to be ASAR by PFA-100 were found to be sensitive in TEG and aggregometer assays when using AA as the agonist. Furthermore, some participants showed a gain of platelet function following ASA when studied on the TEG and aggregometer using ADP as the agonist. Currently, it is unclear which response to ASA treatment is most important to predict cardiovascular complications in normal individuals or patients placed on aspirin. Gum et al, showed that increased urinary secrection of thromboxane metabolites, suggesting ASA insensitivity, is associated with a higher incidence of cardiovascular events. Unfortunately, this test may simply indicate poor patient compliance rather than true ASAR, so a clear demonstration of platelet sensitivity will also be necessary. Our data confirms the need for a collaborative trial comparing different platelet assays to assess true ASA sensitivity together with concurrent measurements of urinary thromboxane metabolite levels. Knowing which assay best links aspirin sensitivity to disease outcome will allow physicians to better manage normal individuals and patients at risk for significant cardiovascular disease.

Cyclooxygenase-2 and an early stage of chronic hypoxia-induced pulmonary hypertension in newborn pigs

Our objective was to determine whether cyclooxygenase (COX)-2-dependent metabolites contribute to the altered pulmonary vascular responses that manifest in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the COX-2 selective inhibitor NS-398 on responses to arachidonic acid or acetylcholine (ACh) was measured in endothelium-intact and denuded pulmonary arteries (100- to 400-microm diameter). Pulmonary arterial production of the stable metabolites of thromboxane and prostacyclin was assessed in the presence and absence of NS-398. Dilation to arachidonic acid was greater for intact control than for intact hypoxic arteries, was unchanged by NS-398 in intact arteries of either group, and was augmented by NS-398 in denuded hypoxic arteries. ACh responses, which were dilation in intact control arteries but constriction in intact and denuded hypoxic arteries, were diminished by NS-398 treatment of all arteries. NS-398 reduced prostacyclin production by control pulmonary arteries and reduced thromboxane production by hypoxic pulmonary arteries. COX-2-dependent contracting factors, such as thromboxane, contribute to aberrant pulmonary arterial responses in piglets exposed to 3 days of hypoxia.

Effects of thiol antioxidant on Reduced Nicotinamide Adenine Dinucleotide Phosphate oxidase in hypertensive Dahl salt-sensitive rats

Recent studies implicate of reactive oxygen species (ROS) in hypertension; however, whether reactive oxygen species promote hypertensive derangements is not fully clear. We thus investigated the effects of an antioxidant, n -acetyl- l-cysteine, on hypertensive Dahl salt-sensitive rats. High-salt intake for 4 weeks markedly elevated systolic arterial pressure, urinary excretion of protein, 8-isoprostane, and H 2O 2, and the enzyme activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase along with the elevated expression of its subunits gp91phox and p47phox at the levels of mRNA and protein. Supplement with n -acetyl- l-cysteine reduced the increase in systolic arterial pressure and counteracted the elevation of urinary excretion of protein, 8-isoprostane, and H 2O 2, and the increases in NADPH oxidase activity/expression in high-salt-loaded Dahl salt-sensitive rats. n -acetyl- l-cysteine supplement ameliorated plasma and urinary levels of thromboxane B 2 (an end metabolite of thromboxane A 2), associated with improvement of both the abnormal contraction and the impaired nitric oxide-dependent relaxation in renal arteries. These results revealed that oxidative stress mediates hypertensive changes in Dahl salt-sensitive rats, because thiol antioxidant n -acetyl- l-cysteine attenuated the augmentation of local ROS production by diminishing the elevation of NADPH oxidase expression and ameliorated renal/vascular hypertensive changes.

The antithrombotic profile of aspirin. Aspirin resistance, or simply failure?

The antithrombotic profile of aspirin. Aspirin resistance, or simply failure?

Eicosanoids in preeclampsia

Preeclampsia is characterized by an imbalance between two cyclooxygenase metabolites of arachidonic acid, thromboxane and prostacyclin, that favors thromboxane. Because of the biologic actions of these two eicosanoids, this imbalance might explain major clinical symptoms of preeclampsia, such as hypertension, platelet aggregation and reduced uteroplacental blood flow. In the maternal circulation, this imbalance is primarily manifested by decreased production of prostacyclin by endothelial cells. Platelet thromboxane synthesis is only increased in severe preeclampsia. In the placenta and in leukocytes, the imbalance is exacerbated by increased production of thromboxane coupled with decreased production of prostacyclin in both mild and severe preeclampsia. Longitudinal measurements of urinary metabolites of thromboxane and prostacyclin reveal that the thromboxane/prostacyclin imbalance predates the onset of clinical symptoms of preeclampsia. The imbalance between thromboxane and prostacyclin is most likely caused by oxidative stress, which is manifest in preeclampsia by increased lipid peroxidation and decreased antioxidant protection. Oxidative stress may drive this imbalance because lipid peroxides activate the cyclooxygenase enzyme to increase thromboxane synthesis, but at the same time they inhibit prostacyclin synthase to decrease prostacyclin synthesis. Low-dose aspirin therapy (50–150 mg/day) has been considered for the prevention of preeclampsia because it selectively inhibits thromboxane synthesis. Several studies reported dramatic decreases in the incidence of preeclampsia with low-dose aspirin therapy. However, two large multicenter studies reported only modest decreases, which dampened enthusiasm. The two large studies were “intent to treat” studies which included patients who were noncompliant and who discontinued the use of aspirin. In one of the studies for which compliance statistics were available only 53% of the aspirin group had a compliance rate greater than 75%, which raises a question as to whether the effectiveness of aspirin was being tested. Low-dose aspirin therapy should not yet be dismissed for the prevention of preeclampsia, but be reconsidered with emphasis on compliance using doses of aspirin in the range of 100–150 mg/day combined with antioxidants.

Cyclooxygenase isoforms and platelet vessel wall interactions in the apolipoprotein E knockout mouse model of atherosclerosis.

Cyclooxygenase (COX) activity is induced in human atherosclerosis, and the products formed may modify the disease directly or through an effect on platelets. We examined the role of COX-1 and -2 on platelet vessel wall interactions and development of atherosclerosis in a murine model. Apolipoprotein E-deficient (apoE-/-) mice fed a 1% cholesterol diet were treated with a selective COX-1 inhibitor (SC-560), a selective COX-2 inhibitor (SC-236), or vehicle. Urinary prostacyclin and thromboxane metabolites (2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-TXB2) were increased in the apoE-/- knockout mouse. There was also induction of both COX isoforms in the vascular lesions formed, which stained for CD41, a platelet-specific marker, and for CD40L. Selective inhibition of COX-2 had no effect on lesion formation and, despite selective reduction in prostacyclin generation, had no effect on platelet activity, as measured by thromboxane formation or platelet deposition. Selective inhibition of COX-1 reduced 2,3-dinor-TXB2 generation and lesion formation. However, platelet deposition on the vessel wall persisted, with well-defined monolayers seen. There was also persistent expression of the macrophage marker CD68 and increased expression of the cell death protein Bax. In contrast to lesion development, the selective COX-1 inhibitor had no effect on the regression of evolving lesions. COX-1 plays an important role in the early stages of lesion development in the apoE-/- knockout model of atherosclerosis, preventing gross lesion formation in the face of continued vascular injury and inflammation. Despite the inhibition of prostacyclin, COX-2 inhibition had no effect on lesion development or platelet-vessel wall interactions.

Cyclooxygenase-2 Expression and Inhibition in Atherothrombosis

Arachidonic acid metabolism plays an important role in acute ischemic syndromes affecting the coronary or cerebrovascular territory, as reflected by biochemical measurements of eicosanoid biosynthesis and the results of inhibitor trials in these settings. Two cyclooxygenase (COX)-isozymes have been characterized, COX-1 and COX-2, that differ in terms of regulatory mechanisms of expression, tissue distribution, substrate specificity, preferential coupling to upstream and downstream enzymes, and susceptibility to inhibition by the extremely heterogeneous class of COX-inhibitors. Although the role of platelet COX-1 in acute coronary syndromes and ischemic stroke is firmly established through approximately 20 years of thromboxane metabolite measurements and aspirin trials, the role of COX-2 expression and inhibition in atherothrombosis is substantially uncertain, because the enzyme was first characterized in 1991 and selective COX-2 inhibitors became commercially available only in 1998. In this review, we discuss the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque "vulnerability," and the clinical consequences of COX-2 inhibition. Recent studies from our group suggest that variable expression of upstream and downstream enzymes in the prostanoid biosynthetic cascade may represent important determinants of the functional consequences of COX-2 expression and inhibition in different clinical settings.

The effects of a cyclo-oxygenase II inhibitor on placental artery production of thromboxane and prostacyclin

Objective The study was undertaken to determine the effects of a cyclo-oxygenase II inhibitor on fetoplacental artery production of prostacyclin and thromboxane A 2. Study design Eight placentas were obtained from normal parturients at delivery and four chorionic plate arteries were dissected from each placenta. Arteries were incubated in media alone, media plus angiotensin II (1×10 −10 mol), media plus rofecoxib (300 ng/mL), or media plus angiotensin II and rofecoxib. Serial samples were assayed for metabolites of thromboxane B 2 and prostacyclin by enzyme-linked immunosorbent assay. Results were compared by analysis of variance, and P<.05 was considered significant. Results At 24 hours, 6-keto-prostaglandin F 1α levels in the rofecoxib group (1.74±1.39 ng/mg tissue, P<.01) and the rofecoxib plus angiotensin II group (2.15±1.85 ng/mg tissue, P<.01) were significantly lower than levels in the control group (4.25±2.03 ng/mg tissue). Thromboxane B 2 levels were lower in the angiotensin II group (0.65±0.33 ng/mg tissue) than the control group (1.22±0.70 ng/mg tissue, P<.05). Conclusion Cyclo-oxygenase II inhibition decreases the production of prostacyclin in fetoplacental arteries and alters the normal ratio of thromboxane A 2 to prostacyclin.

Zileuton, a 5-lipoxygenase inhibitor, increases production of thromboxane A2 and platelet aggregation in patients with asthma.

Leukotrienes, generated from arachidonic acid via the lipoxygenase pathway, play an important role in the pathophysiology of asthma. Therefore, leukotriene inhibitors, such as Zileuton, are used in the treatment of asthma. However, thromboxanes, generated from arachidonic acid via the cyclooxygenase pathway, play an important role in platelet aggregation and thrombosis. Therefore, we studied whether Zileuton, by shifting arachidonic acid to the cyclooxygenase pathway, enhances thromboxane production and, hence, platelet aggregation. Blood samples were collected from 10 asthmatic patients before and 2 weeks after standard Zileuton treatment. Spontaneous platelet aggregation was measured in platelet-rich plasma. Platelet-rich plasma was also used to determine thromboxane B(2), a stable metabolite of thromboxane A(2), as the indirect measure of thromboxane A(2) because thromboxane A(2) is too unstable for assay. Baseline thromboxane B(2) and platelet aggregation values in the 10 asthmatic patients were normal. Treatment with Zileuton for 2 weeks significantly increased thromboxane B(2) levels from baseline levels of 267 +/- 54 microg/l to 389 +/- 62 microg/l after 2 weeks of treatment (P < 0.0002). Spontaneous platelet aggregation also increased significantly from baseline values of 4.2 +/- 2.4% to 6.8 +/- 2.8% after 2 weeks of treatment (P < 0.0001). These results establish that Zileuton, an effective drug for asthma, adversely affects in vitro platelet function. The findings suggest that this drug, and perhaps related agents also, may pose a thrombotic risk; clinical attention will be needed to address this possibility.

Aspirin and clopidogrel in acute coronary syndromes: therapeutic insights from the CURE study.

Platelet adhesion, activation, and aggregation are central to thrombus formation, which follows atherosclerotic plaque disruption and causes acute coronary syndromes. Aspirin and clopidogrel exert their antiplatelet effects by inhibiting thromboxane A2 production and adenosine diphosphate-induced platelet aggregation pathways, respectively. Aspirin has proven benefits in primary and secondary prevention of coronary artery disease. Clopidogrel, an alternative antiplatelet agent used in patients with aspirin intolerance, is especially useful in combination with aspirin after coronary stent procedures. The CURE (Clopidogrel in Unstable Angina to Prevent Recurrent Events) study demonstrates for the first time the benefit of adding clopidogrel to aspirin rather than using aspirin alone in patients having acute coronary syndromes without ST-segment elevation myocardial infarction. Patients who are resistant to aspirin (up to 10%) have higher rates of cardiovascular events and may derive special benefit from the combination therapy. Aspirin resistance can be assessed through platelet aggregometry testing, measurement of urinary thromboxane metabolites, and, possibly, genomic testing in the future.

A thromboxane A 2 system in the Atlantic stingray, Dasyatis sabina

Thromboxane B 2 ( TXB 2) is the stable metabolite of thromboxane A 2 ( TXA 2) and thromboxane B 2-like immunoreactivity (iTXB 2) has been identified in the plasma of the Atlantic stingray, Dasyatis sabina (0.57±0.03 ng/ml). Plasma levels of iTXB 2 increase if the blood is allowed to clot (3.0±0.27 ng/ml). When clotting occurs in the presence of indomethacin, this increase is partially inhibited (1.5±0.17 ng/ml), indicating the presence of a cyclooxygenase activity. Radioligand binding analysis using the TXA 2 analog [ 125 I ]BOP in isolated kidney membranes revealed a receptor of K d=2.88±0.51 nM and B max=25.6±5.9 fmol/mg protein. [ 125 I ]BOP binding was displaced by the TXA 2 receptor (TP receptor) agonists U46619 (IC 50=106.4±15.7 nM) and U44069 (IC 50=88.7±13.0 nM), and the antagonist SQ29548 (IC 50=51.0±12.9 nM). Binding was also displaced stereoselectively by the antagonists (−)L657925 (IC 50=18.9±3.8 nM) and (+)L657926 (IC 50=2025±280 nM). Tissue bath studies revealed that U46619, a stable TXA 2 mimetic, elicited concentration-dependent contractions in the ventral aorta which were inhibited in a concentration-dependent manner by the TP receptor antagonist SQ29548. Using a human TP receptor riboprobe, Northern blotting of mRNA isolated from the stingray kidney identified transcripts of 2.8 and 6 kb. The 2.8 kb transcript is similar to a 2.8 kb transcript found in human cells or tissues, but the 6 kb transcript may be unique. These data indicate the presence of a TXB 2-like substance in the blood, a TP receptor in the kidney, TXA 2 biological activity in the ventral aorta, and expression of a TP receptor-like gene.

Cocaine decreases urinary prostacyclin release in pregnancy: correlation with uterine and umbilical Doppler velocimetry

Objective: The purpose of this study was to determine maternal urinary prostacyclin and thromboxane excretion in patients with recent cocaine use, compared to cocaine-free controls, and correlate the findings with Doppler velocimetry.Study design: Seventeen patients admitted withpremature rupture of membranes between the gestational ages of 24 and 34 weeks were tested for urinary cocaine metabolites. Eleven patients had positive screening and six patients were negative and served as controls. After initial stabilization, 24-hurine collections were obtained and were assayed for the prostacyclin metabolite 2,3-dinor-6-keto-PGF1α, and the thromboxane metabolite 2,3-dinor TXB2. These patients underwent uterine and umbilical arterial Doppler velocimetry expressed as the pulsatility index. Statistical analysis was performed by the SPSS statistical package using the non-paired Student ttest, and Spearman correlation coefficient with p < 0.05 being considered significant.Results: Compared withcontrols, urinary excretion of prostacyclin in pregnant women with recent cocaine use was significantly lower. The pulsatility index of the uterine arteries of the cocaine-positive individuals was higher than in controls and had a significant inverse correlation withprostacyclin metabolite excretion. Umbilical arterial velocimetry was similar in the two groups.Conclusion: Recent cocaine ingestion in pregnant women decreases production of prostacyclin and negatively affects the pulsatility index of the uterine artery.

NCX-4016 (NO-aspirin) inhibits lipopolysaccharide-induced tissue factor expression in vivo: role of nitric oxide.

NCX-4016 is an acetylsalicylic acid (ASA) derivative containing a nitric oxide-releasing moiety. Compared with ASA, NCX-4016 has a broader spectrum of antithrombotic and antiinflammatory activities. We hypothesized that NCX-4016 might inhibit in vivo lipopolysaccharide (LPS)-induced expression of tissue factor (TF). Rats were administered 90 mg/kg NCX-4016 orally for 5 days. Placebo, 50 mg/kg ASA, and 80 mg/kg isosorbide-5-mononitrate (ISMN) were used in control groups. On day 5, rats were injected intraperitoneally with 100 microg/kg LPS and killed 6 hours later. The expression of TF in monocytes was measured by flow cytometry and Western blot analysis. Reverse transcriptase-polymerase chain reaction was performed to assess expression of TF and cyclooxygenase-2 (COX-2) genes. Plasma concentrations of interleukin-1beta and tumor necrosis factor-alpha were measured. Urine samples were collected to evaluate the excretion of the thromboxane metabolite 11-dehydro-thromboxane (TX)B2. Gastric mucosa was inspected. LPS injection was followed by synthesis TF and COX-2 mRNAs in circulating monocytes, which were blunted by NCX-4016 but not by ASA or ISMN. Both NCX-4016 and ISMN reduced TF expression on surface of circulating monocyte. LPS increased the excretion 11-dehydro-TXB2, and this was prevented by NCX-4016 and ASA. Unlike ASA, NCX-4016 reduced plasma interleukin-1beta and tumor necrosis factor-alpha. In addition, NCX-4016 almost completely prevented mucosal damage, whereas ASA increased the extension of gastric lesions in LPS-injected rats. NCX-4016 prevents monocyte TF expression; this is accompanied by inhibition of TX and cytokine biosynthesis. These additive effects of nitric oxide release and COX inhibition may help explain efficacy and tolerability of NCX-4016.

Effect of the ingestion of Ginkgo biloba extract on platelet aggregation and urinary prostanoid excretion in healthy and Type 2 diabetic subjects

Enhanced platelet function, particularly in response to collagen, is a common occurrence in diabetes that increases the risk of cardiovascular disease. Ginkgo biloba extract is ingested primarily to improve mental focus but it possesses a blood-thinning potential, which has not been well characterized. This study was designed to compare the effect of ingesting G. biloba extract on platelet aggregation in platelet-rich plasma (PRP) and prostanoid urinary excretion in healthy volunteers and subjects with Type 2 diabetes mellitus (T2DM). Before and after ingesting 120 mg of standardized G. biloba extract for 3 months, platelet aggregation was studied in PRP and urinary metabolites of thromboxane B 2 (TXB 2) and prostacyclin (PGI 2) were measured. In healthy volunteers (age, 42±11 years; BMI, 28.4±4.8 kg/m 2; n=28), the ingestion of G. biloba extract significantly increased fasting insulin and C-peptide (10±4 vs. 12±6 μU/ml, p<0.007 and 1.3±0.8 vs. 2.1±1.1 ng/ml, p<0.001, respectively) and significantly reduced collagen but not PAF-mediated platelet aggregation, converting 21 of 28 subjects with [COL+/EPI+] platelets to the [COL−/EPI+] phenotype. This was accompanied by a reduction of 11-dehydro-TXB 2 from 12.4±6.1 to 10.3±6.1 ng/mg Cr ( p<0.04) and PGI 2 metabolites (2,3-dinor-6-keto-PGF 1α and 6-keto-PGF 1α) from 2.2±0.8 to 1.8±0.8 ng/mg Cr ( p<0.05). In the T2DM subjects (age, 54±8; BMI, 36.6±7.9 kg/m 2; n=19), G. biloba ingestion did not affect pancreatic β-cell function but significantly reduced platelet aggregation, converting 16 of 19 [COL+/EPI+] platelets to the [COL−/EPI+] phenotype. Unlike the healthy volunteers, this was not accompanied by a reduced urinary prostanoid excretion. G. biloba-induced reduction of both classes of prostanoid metabolites in healthy volunteers, but not in T2DM subjects, may suggest a nonselective inhibition of COX-1-mediated TXA 2 in platelets and COX-2-mediated PGI 2 production by the endothelial cells and perhaps platelet-enriched levels of arachidonic acid or COX-1 activity, or both, in T2DM subjects.

Hypertension Plus Diabetes Mimics the Cardiomyopathy Induced by Nitric Oxide Inhibition in Rats

We compared the myocardial lesions caused by the long-term inhibition of nitric oxide (NO) biosynthesis with those associated with renovascular hypertension (two-kidney, one-clip model [2K-1C]) and superimposed streptozotocin-induced diabetes mellitus (DM). Prospective trial. University laboratory. Male Wistar rats were classified into the following groups: (1) a control group; (2) the L-NAME group (treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester [L-NAME], 75 micro mol per rat per day, orally); (3) the 2K-1C group (renovascular hypertension); (4) the DM group (treatment with streptozotocin, 60 mg/kg via intraperitoneal route); and (5) the 2K-1C plus DM group (renovascular hypertension and streptozotocin-induced DM). Arterial BP was measured by a tail-cuff method for 3 weeks, after which histologic and stereological analysis of the heart was done and cardiac NO synthase type 3 (NOS3) levels were assessed by Western blotting. The circulating levels of nitrates/nitrites and thromboxane B(2) (TXB(2), the stable metabolite of thromboxane A(2)) were also measured. In DM and 2K-1C rats, the myocardial lesions consisted mainly of recent myocardial infarcts, which were more severe in the 2K-1C plus DM group. In L-NAME-treated rats, multiple foci of reparative fibrosis and fresh myocardial necrosis resembled the severe lesions found in the 2K-1C plus DM group. Although NOS3 protein expression increased (19 to 44%; p < 0.05) in all treated groups, serum nitrate/nitrite levels decreased only in the L-NAME group and the 2K-1C plus DM group. These two groups also showed a more pronounced increase in TXB(2) concentrations. These results indicate that the association of hypertension and DM mimics the alterations induced by L-NAME in rats, which suggests a role for NO in the pathophysiology of hypertensive-diabetic cardiomyopathy.

Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs.

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100-400-microm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100-400-microm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic. Radioimmunoassay was used to determine pulmonary artery production of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and were similar for both groups. 6-Keto-PGF(1alpha) production was reduced, whereas TxB(2) production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Effects of the thromboxane synthetase inhibitor and receptor antagonist terbogrel in patients with primary pulmonary hypertension

Background Circulating mediators, including thromboxane A2, the vasoconstrictor, platelet aggregant, and smooth muscle mitogen, may contribute to the progression of vascular narrowing in primary pulmonary hypertension (PPH). Methods To further understand the contribution of thromboxane and to provide novel therapy for PPH, we administered the potent orally active thromboxane synthetase inhibitor and thromboxane receptor antagonist terbogrel for 12 weeks to patients with New York Heart Association functional classification II and III PPH. The study had a multicenter randomized placebo-controlled design. The primary endpoint was a change in the distance walked during 6 minutes. The pharmacologic effects of terbogrel on thromboxane and prostacyclin metabolism also were studied. Results Although the planned enrollment was 135 patients, the study was halted after only 71 patients had been randomized because of the unforeseen side effect of leg pain, which occurred almost exclusively in patients with terbogrel treatment. Only 52 patients completed the 12-week study, and only 22 patients (31%) were fully compliant with the study medication. The leg pain confounded the primary endpoint of walking distance. On an intention-to-treat analysis, no improvements in 6-minute walk distance or in hemodynamics in patients with terbogrel treatment were seen. However, terbogrel was effective from a pharmacologic standpoint, reducing thromboxane metabolites by as much as 98% (P <.0001), with a modest but statistically insignificant (39%) rise in prostacyclin metabolites. Conclusion Inhibition of thromboxane with an orally active agent is feasible in PPH, but the incidence of severe leg pain with terbogrel precludes its use in this disorder. Similar therapeutic efforts, with other thromboxane inhibitors, should be considered. (Am Heart J 2002;143:e4)

Oxidative stress-induced isoprostane formation may contribute to aspirin resistance in platelets

Recent studies suggest that aggregation of platelets from patients with coronary artery and cerebrovascular disease may be resistant to low-dose aspirin (ASA) treatment, which may promote plaque-associated thrombus formation. However, the underlying mechanisms of platelet ASA resistance are poorly understood. ASA is thought to inhibit platelet aggregation primarily by inactivating the cyclooxygenase (COX), thus decreasing the synthesis of the pro-aggregatory arachidonic acid metabolite thromboxane A 2 (TxA 2). However, recent studies also identified a non-enzymatic, oxidation-dependent pathway for the synthesis of the arachidonic acid derivative isoprostanes, which exhibit potent vasoconstrictor and pro-aggregatory effects similar to that of TxA 2. Because the pathophysiological conditions that promote arteriosclerotic vascular diseases (e.g. hypercholesterolemia, diabetes, hyperhomocysteinemia) are thought to be associated with an increased formation of reactive oxygen species and increased plasma isoprostane levels, it can be hypothesized that increased COX-independent isoprostane formation in platelets contribute to ASA resistance.