TXB2 Levels Research Articles

This Month in Gastroenterology

This Month in Gastroenterology

Daily treatment with sildenafil, an inhibitor of cGMP-dependent phosphodiesterase, normalizes the exaggerated hemodynamic response to norepinephrine in fructose-fed rats

Background Enhanced responsiveness of the vascular smooth muscle cell to vasoconstrictor stimuli has been described in insulin-resistant states. Fructose overload leads to the development of the metabolic syndrome and its associated cardiovascular disorders including hypertension. We postulated that chronic treatment with sildenafil would regulate the abnormally increased pressor response to a norepinephrine challenge in fructose-fed rats. Material and methods Wistar rats were fed a standard chow (CONT, n = 12) or a 60% fructose-enriched diet containing 5% fat (% by weight) for 8 weeks (FFR, n = 12). From week 5 through 8, sildenafil (twice a day sc, 20 mg/kg) was administered (FFR+SIL, n = 14), thus reaching clinically relevant plasma concentrations circa 20 nM unbound (Pfizer Inc., data on file), followed by a 1-week wash-out period (W9). On W9, in a sub-group of these conscious rats (n = 5) that had previously been implanted with telemetric devices, AP was recorded following cumulative infusion of noradrenaline (NA 50, 100, 200, 400 ng/kg/min). In all rats, urinary 8-isoprostane (IPT) and TxB2 levels were determined during a 24-hour period. Tissue ET-1 content was measured in segments of aortic and mesenteric artery. Results Fasting glycemia was unchanged in all rats while triglyceridemia were elevated in FFR compared to CONT (2.0 ± 0.4 mM vs 1.2 ± 0.2, p < 0.05) and corrected by sildenafil treatment (1.3 ± 0.2, NS vs CONT). Resting AP was similar in all rats. Pressor responses to NA were exacerbated in FFR (maximal increase from basal MAP: +22.8 ± 1.7 vs +38.0 ± 7.3 mmHg, respectively, p < 0.05) and normalized by sildenafil treatment (+24.9 ± 5.2 mmHg, NS vs CONT). Vascular ET-1 levels were not modified by the fructose nor sildenafil treatment whereas the increased production of urinary IPT and TxB2 in FFR versus CONT (2.07 ± 0.36 ng/ml/24 h vs 0.88 ± 0.13 and 12.86 ± 1.91 pg/ml/24 h vs 7.25 ± 0.59, respectively, p < 0.05) were corrected following sildenafil treatment (0.95 ± 0.14 ng/ ml/24 h and 8.74 ± 1.06 pg/ml/24 h, respectively, NS vs CONT).

Invited commentary

The authors’ manuscript reflects an area of growing interest in other specialties and should serve to stimulate research opportunities in vascular surgery. Articles relating to aspirin resistance have been published in the stroke and cardiology literature, where an increasing number of studies have shown an association between the incidence of adverse thrombotic events and aspirin resistance. The subject, however, has not been widely addressed in the vascular literature. While important, the manuscript must be taken in context. As noted by the authors, aspirin resistance is not yet clearly defined, and can be characterized by laboratory measures or adverse clinical events. Laboratory measures of platelet function can include in vivo testing, such as bleeding time. Ex vivo tests include the measurement of arachidonic acid metabolites, such as urinary 11-dehydro TXB2; measures of platelet-platelet interaction, such as optical or impedance aggregometry; point of care cartridge based analyzers, such as the PFA-100; and whole blood flow cytometry, which measures activation dependent changes on the platelet surface (such as CD62p, p-selectin; activated GPIIb/IIIa; leukocyte platelet aggregates), activation dependent signaling, and activation dependent release from platelets. Of concern, however, the reported incidence of aspirin resistance is variable, even using comparable techniques; resistance in studies employing aggregometry has ranged from 0.4% to 9%, and using the PFA-100 from 9% to 35%.1Dalen J.E. Aspirin resistance: Is it real? Is it clinically significant?.Am J Med. 2007; 120: 1-4Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Clinical studies have demonstrated a higher incidence of aspirin resistance among patients with a prior history of transient ischemic attack or stroke; high risk patients with a history of coronary artery disease have increased odds of being aspirin resistant.2Mason P.J. Jacobs A.K. Freedman J.E. Aspirin resistance and atherothrombotic disease.J Am Coll Cardiol. 2005; 46: 986-993Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Mueller et al.3Mueller M.R. Salat A. Stangl P. Murabito M. Pulaki S. Boehm D. et al.Variable platelet response to low-dose ASA and the risk of limb deterioration in patients submitted to peripheral arterial angioplasty.Thromb Haemost. 1997; 78: 1003-1007PubMed Google Scholar followed 100 patients with intermittent claudication, treated with 100 mg of aspirin per day, for 18 months after balloon angioplasty. Only 40% of male patients showed the expected effect of aspirin using in vitro platelet aggregation. All reocclusions at the site of angioplasty occurred in this group. Higher vascular event rates have also been correlated with measures of aspirin resistance in several prospective studies. Aggregometry, urinary 11-dehydro TXB2 levels, and the PFA-100 have been used to assess platelet function. Interestingly, results from the PFA-100 did not correlate with aggregometry and did not correlate with vascular events.1Dalen J.E. Aspirin resistance: Is it real? Is it clinically significant?.Am J Med. 2007; 120: 1-4Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The current study uses two techniques to assess laboratory evidence of aspirin resistance in patients on chronic aspirin therapy and illustrates the caution that must be exercised in interpreting results. Measurement of arachidonic acid induced expression of CD62p (P-selectin), using flow cytometry, demonstrates that all patients receiving aspirin show an aspirin effect, while PFA-100 closure times indicate 16% of patients taking aspirin show no effect of aspirin. The PFA-100 can be influenced by multiple variables, including platelet count, platelet function, red blood cells, and von Willebrand factor; and the outcome can be effected by variables, such as von Willebrand factor, that are not influenced by aspirin. None-the-less, the current study is a very nice introduction to the concept of aspirin resistance and provides an example of its application in vascular surgery. While the study demonstrates evidence of laboratory resistance, it has failed to exclude patient noncompliance as a source of “resistance” and has not correlated laboratory resistance with clinical evidence of adverse events. The study also demonstrates that laboratory measures can influence the extent and character of laboratory defined resistance. Future studies could randomize aspirin therapy and laboratory adjusted aspirin therapy, based on identified resistance; verification of compliance and documentation of clinical outcomes should be incorporated.

Phenotypic predictors of increased urinary 11-dehydrothromboxane B2 levels in patients treated with aspirin

Inhibition of thromboxane A2 by aspirin can be estimated by urinary levels of a metabolite 11-dehydrothromboxane B2 (TXB2). Our objective was to compare TXB2 levels in relation to cardiovascular risk factors that may affect resistance to aspirin.

Mast Cell Deficiency Leads to Decreased Expression of Cyclooxygenase-1 (COX1), COX2, Toll-Like Receptor-4 (TLR4) and Endothelial Nitric Oxide Synthase (eNOS) mRNA in Mouse Aorta (100.8)

Abstract Mast cells (MC) are well known for their contribution to vascular inflammation and atherosclerosis. We have shown that MC proteases and histamine amplify lipopolysaccharide (LPS)-induced inflammatory responses in human coronary artery endothelial cells via stimulation of TLR4 expression. This study examined mRNA levels of COX1, COX2, TLR4 and eNOS in aortic tissues of MC-deficient (W/Wv) mice and wild type controls (WT) to gain insight into the role of MC in vascular pathophysiology. We also determined the effect of MC degranulation on gene expression in aortas 24 h after intraperitoneal injection of MC degranulation agent compound 48/80 (1 μg/g body wt). We found that, in comparison to WT, aortic tissues of W/Wv mice express lower levels of COX1, COX2, TLR4 and eNOS mRNA. Injection of compound 48/80 resulted in marked stimulation of expression of these genes in aortas of WT, but not in W/Wv mice, confirming the involvement of MC degranulation products in this process. To determine whether a deficiency of MC and/or decreased COX expression in W/Wv mice is associated with changes in prostanoid homoeostasis, the levels of prostacyclin and thromboxane A2 metabolites in urine samples were analyzed. The levels of 6-keto PGF1α and 11-dehydro TXB2 in 24 h urine of both genotypes were comparable when normalized for the volume and creatinine excretion. These results suggest that, although MC deficiency suppresses the expression of COX1 and COX2 genes in the vasculature, the systemic prostanoid homeostasis remains protected. (Supported by NIH R01-HL070101 and Carey Arthritis Fund)

Enhancing Effect of Collagen Receptor Polymorphisms on In Vitro Platelet Reactivity to Aspirin in Healthy Subjects.

[Background]Aspirin (ASA) is widely used as an antiplatelet drug, and a large number of clinical trials with ASA demonstrated significant efficacies for prevention and treatment of athrothrombosis. Recently, accumulating evidences indicated that there are inter-individual variations in the platelet response to ASA. The subpopulation, called ASA resistance, has the inability of response to ASA on ex vivo or in vitro platelet function tests and the poor clinical outcomes, although the mechanism underlying the variability is largely unknown. To date, genetic factors were showed to have an impact on platelet reactivity to ASA, and the inter-individual variations in platelet response to ASA was also reported to be associated with platelet sensitivity to collagen. In this study, the association between collagen-induced platelet aggregation (CIPA) and genetic polymorphisms of collagen receptors, glycoprotein (GP) Ia and GPVI, was analyzed using platelets treated by ASA (ASA +/−). We also investigated the effect of these polymorphisms on platelet thromboxane (TXB2) levels, closely related to the final stages of the arachidonate pathway inhibited by ASA.[Methods]We recruited genetically unrelated Japanese males (n=172) at their regular checkups. The mean age was 46.7±5.1 years. The subjects had no apparent hematologic or vascular disease and were not taking any medications that affect platelet function. Written informed consent was obtained from all study subjects. Platelet-rich plasma (PRP) sample was incubated with ASA [final concentration (fc) 10μM] or vehicle for 30 min at 24 degree Centigrade, and CIPA (fc 2μg/ml) test was performed on each PRP sample. Subsequently, platelet TXB2 levels were measured in the supernatant after centrifugation of each sample of CIPA test. Genotypes of the 807TC, Glu534Lys, Asn927Ser polymorphisms of GPIa and the Ser219Pro, Lys237Glu, Thr249Ala, Gln317Leu, His322Asn polymorphisms of GPVI were determined using the single-nucleotide primer extension-based method.[Results]To examine the sensitivity of platelets to ASA in vitro, we analyzed CIPA and platelet TXB2 levels in ASA(+/−). The maximum platelet aggregation and TXB2 levels in ASA(+) were significantly lower than those in ASA(−) (paired t-test, p<0.0001 and p<0.0001, respectively). Next, we investigated the association between the collagen receptor polymorphisms and the maximum platelet aggregation in ASA (+/−). For ASA(−), all genotypes of GPIa and GPVI were not associated with the maximum platelet aggregation. For ASA(+), subjects with 807TT/TC of GPIa had higher aggregation compared to those with 807CC(P=0.0135) whereas no association was observed between other polymorphisms and the maximum platelet aggregation. Moreover, repeated measures ANOVA showed that the difference in this inhibitory effect of ASA was significant between the 807TT/TC and 807CC genotypes (p=0.0253); the 807CC genotype has higher inhibitory effect of ASA. There was no association between platelet TXB2 levels and the GPIa and GPVI polymorphisms both in ASA(+) and ASA(−).[Conclusion]The 807CC genotype of GPIa polymorphism is associated with higher sensitivity to ASA in CIPA.

Cross Reaction of Antibody Against Helicobacter Pylori Urease B with Platelet Glycoprotein IIIa and Its Significance in the Pathogenesis of ITP.

The data about association of Idiopathic Thrombocytopenic Purpura (ITP) with Helicobacter pylori (H pylori) infection are always contradictory, and the factors that incite ITP remain uncertain. Several hypotheses have been suggested about the striking discrepancies between the reported results, one of which is variable effects of genetically diverse H pylori strains in remote geographic regions. The sequence of gene for H pylori urease B is relatively constant, and its protein production is believed to be a crucial factor resulting in some diseases. We therefore conducted a study to assess the role of H pylori in the pathogenesis of ITP, and to investigate its immune mechanisms. Firstly, bacteria H pylori were isolated from gastric mucosal tissue of a patient with peptic ulcer and cultured. H pylori urease B gene was amplified, then ligated into expression plasmid. The latter was transfected into E coli. The recombinant urease B was used to develop monoclonal antibody (MoAb), 1F11, against urease B of H pylori. In Western blot analysis, both 1F11 and SZ21 (MoAb against GP IIIa) bound to the band of protein 95 KDa of normal platelet lysate, but not to that of platelet lysate prepared from a patient with Glanzmann thrombobasthenia (GT), in whom platelet membrane GP IIb/IIIa were absent. Flow cytometry analysis also showed that normal platelets were reacted with both 1F11 and SZ21, whereas platelets of the GT patient showed a low reactivity with 1F11 or SZ21. In immunoradiometric assay, in which four MoAbs against different platelet membrane GPs (GP Ib, GP IIb, GP IIIa and GP IV) were each coated on microtiter plates, and platelet lysate and then 125I-labeled 1F11 were separately added, the radioactivity value (count per minute) in plates coated with SZ21 was the highest among all coated plates. All the three different experimental results showed that there exists a cross reaction between MoAb 1F11 against Helicobacter pylori urease B and platelet glycoprotein IIIa. In addition, MoAb 1F11 could inhibit ADP induced platelet aggregation in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 levels induced by ADP, because platelet P-selectin expression and TXB2 synthesis could occur before aggregation, the “final step” of platelet aggregation, just like what reported in Glanzmann thrombocytopenia. Finally, we performed a prospective clinical study on H pylori infection by means of immuno-radiometric assay of urease B in 164 ITP patients, 88 (54%) were found H pylori-positive. platelet counts in 15 of 38 H pylori-positive patients were significantly increased after eradication treatment, and were completely recovered in 7 patients. In conclusion, our results first indicate that antibidy against H pylori urease B could cross-react with platelet GP IIIa, and inhibit platelet aggregation. H pylori eradication may improve the platelet counts in some chronic ITP patients.

Influence of L-arginine on the expression of eNOS and COX2 in experimental pulmonary thromboembolism

The influence of L-arginine on endothelial nitric oxide synthase (eNOS) and cyclooxygenase 2 (COX2) was observed in experimental pulmonary thromboembolism and the action mechanism on pulmonary thromboembolism was explored. Wistar rats were randomly divided into control group, model group and treatment group. Pulmonary thromboembolism models were established by auto-blood back transfusion, and L-Arg 100 mg/kg was intraperitoneally injected after successful model preparation. The animals were sacrificed at 3 h, 1 day, 3 days and 7 days after embolism. Plasma NO, TXB2 and 6-Keto-PGFla were detected. The expression of eNOS and COX2 protein and mRNA in pulmonary tissues was detected by immunohistochemistry and RT-PCR respectively. The results showed that pulmonary thrombosis could be seen post pulmonary embolism and inflammatory reaction was significant. Plasma NO was decreased (P<0.01), and the levels of TXB2, 6-Keto-PGF1alpha and T/P ratio were all elevated. The expression of eNOS protein and mRNA in the pulmonary tissue was down-regulated (P<0.05), while that of COX2 protein and mRNA was upregulated (P<0.01). In treatment group, the level of NO was increased, the levels of TXB2 and T/P ratio were decreased, but the level of 6-Keto-PGF1alpha was increased. The expression of eNOS protein and mRNA in pulmonary tissue was upregulated (P<0.05), while that of COX2 protein and mRNA was down-regulated (P<0.05). In conclusion, L-arginine can educe the role of pulmonary tissue protection through up-regulating the expression of intra-pulmonary NOS and down -regulating COX2 in pulmonary thromboembolism.

Heterogeneity in the suppression of platelet cyclooxygenase-1 activity by aspirin in coronary heart disease

Complete and persistent suppression of platelet thromboxane (TX) A(2) biosynthesis by aspirin is mandatory to fulfill its cardioprotection. We explored the determinants of heterogeneity of TXB2 generation in clotting whole blood, a capacity index of platelet cyclooxygenase (COX) activity, in patients with coronary heart disease (CHD) versus healthy subjects treated with low-dose aspirin on a long-term basis. We studied 30 patients with CHD (ie, chronic stable angina, unstable angina, and acute myocardial infarction) and 10 healthy subjects, who were treated with low-dose aspirin (100 mg daily) on a long-term basis, 12 hours after the administration of 160 mg aspirin to ensure saturation of platelet COX-1 activity. Serum TXB2 levels were assessed. The contribution of blood COX-2 to TXA2 biosynthesis was explored by evaluation of the effect of a selective COX-2 inhibitor (L-745,337) added to heparinized whole blood stimulated with Ca++ ionophore A23187 (20 micromol/L) for 1 hour or lipopolysaccharide (0.1 microg/mL) for 4 hours. In healthy subjects serum TXB2 levels ranged from 0.6 to 7.9 ng/mL (median, 2.1 ng/mL; mean +/- SD, 3.2 +/- 2.6 ng/mL). In CHD patients we detected enhanced variability in serum TXB2 generation (median, 3.1 ng/mL [range, 0.15-47 ng/mL]; mean, 8.5 +/- 12.3 ng/mL), which in 8 patients (27%) exceeded the mean value + 2 SDs detected in healthy subjects (ie, 8.4 ng/mL), set as the limit value for an adequate inhibition of platelet COX-1 by aspirin. Elevated whole-blood TXB2 generation was not dependent on leukocyte count, COX-2 activity, or cigarette smoking but was plausibly a result of defective suppression of platelet COX-1 activity. Heterogeneity in the suppression of platelet COX-1 activity by aspirin occurred in CHD patients. The measurement of the serum TXB2 level seems to be an appropriate biomarker to identify patients who have an inadequate inhibition of platelet COX-1 activity by aspirin.

Prostaglandins mediate tonic contraction of the guinea pig and human gallbladder

The gallbladder (GB) maintains tonic contraction modulated by neurohormonal inputs but generated by myogenic mechanisms. The aim of these studies was to examine the role of prostaglandins in the genesis of GB myogenic tension. Muscle strips and cells were treated with prostaglandin agonists, antagonists, cyclooxygenase (COX) inhibitors, and small interference RNA (siRNA). The results show that PGE2, thromboxane A2 (TxA2), and PGF(2alpha) cause a dose-dependent contraction of muscle strips and cells. However, only TxA2 and PGE2 (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and TxB2 (TxA2 stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PGE2 levels. Both inhibitors and the nonselective COX inhibitor indomethacin abolished the tonic contraction. Transfection of human GB muscle strips with COX-1 siRNA partially lowered the tonic contraction and reduced COX-1 protein expression and TxB2 levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PGE2. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, TxB2, and PGE2 levels; a COX-1 inhibitor prevented the increase in tension and TxB2; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PGE2. Indomethacin blocked the rise in tension and TxB2 and PGE2 levels. We conclude that PGE2 generated by COX-2 and TxA2 generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PGE2 and TxA2 levels.

Prostaglandin A1 inhibits increases in intracellular calcium concentration, TXA2 production and platelet activation1

In our previous studies we found that cyclopentenane prostaglandin A1 (PGA1) had neuroprotective effects in a rodent ischemic model. In the present study we aimed to investigate the inhibitory effect of PGA1 on platelet function. The rate of aggregation of human platelets was measured by using turbidimetry. The rate of adhesion of platelets to cultured endothelial cells was determined by using [(3)H]-adenine labeled platelets. 5-Hydroxytryptamine release from platelets was measured with O-phthaldialdehyde fluorospectrophotometry. The levels of TXB(2), a stable metabolite of TXA(2), were determined by radioimmuno-assay. Alternations in platelet morphology were observed using an electron microscope, and the intraplatelet free calcium concentrations were measured with Fluo-3/AM FCM assay. PGA1 significantly inhibited thrombin-, collagen- and ADP-induced aggregation and adhesion of platelets. The morphological changes of platelets induced by thrombin were blocked by PGA1. PGA1 inhibited the release of 5-hydroxytyptamine from dense granules and the synthesis of TXA(2). PGA1 inhibits the activation of platelets probably through blocking increases in intracellular calcium concentration and TXA(2) synthesis.

Biological effects of aspirin and clopidogrel in a randomized cross-over study in 96 healthy volunteers.

Some data suggest that biological 'resistance' to aspirin or clopidogrel may influence clinical outcome. The aim of this study was to evaluate the relationship between aspirin and clopidogrel responsiveness in healthy subjects. Ninety-six healthy subjects were randomly assigned to receive a 1-week course of aspirin 100 mg day(-1) followed by a 1-week course of clopidogrel (300 mg on day 1, then 75 mg day(-1)), or the reverse sequence, separated by a 2-week wash-out period. The drug effects were assessed by means of serum TxB2 assay, platelet aggregation tests, and the PFA -100 and Ultegra RPFA -Verify Now methods. Only one subject had true aspirin resistance, defined as a serum TxB2 level > 80 pg microL(-1) at the end of aspirin administration and confirmed by platelet incubation with aspirin. PFA-100 values were normal in 29% of the subjects after aspirin intake, despite a drastic reduction in TxB2 production; these subjects were considered to have aspirin pseudo-resistance. Clopidogrel responsiveness was not related to aspirin pseudo-resistance. Selected polymorphisms of platelet receptor genes were not associated with either aspirin or clopidogrel responsiveness. In healthy subjects, true aspirin resistance is rare and aspirin pseudo-resistance is not related to clopidogrel responsiveness.

A study of aspirin and clopidogrel in idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is characterised by in situ thrombosis and increased thromboxane (Tx) A2 synthesis; however, there are no studies of antiplatelet therapy in IPAH. The aim of the current study was to determine the biochemical effects of aspirin (ASA) and clopidogrel on platelet function and eicosanoid metabolism in patients with IPAH. A randomised, double-blind, placebo-controlled crossover study of ASA 81 mg once daily and clopidogrel 75 mg once daily was performed. Plasma P-selectin levels and aggregometry were measured after exposure to adenosine diphosphate, arachidonic acid and collagen. Serum levels of TxB2 and urinary metabolites of TxA2 and prostaglandin I2 (Tx-M and PGI-M, respectively) were assessed. A total of 19 IPAH patients were enrolled, of whom nine were being treated with continuous intravenous epoprostenol. ASA and clopidogrel significantly reduced platelet aggregation to arachidonic acid and adenosine diphosphate, respectively. ASA significantly decreased serum TxB2, urinary Tx-M levels and the Tx-M/PGI-M ratio, whereas clopidogrel had no effect on eicosanoid levels. Neither drug significantly lowered plasma P-selectin levels. Epoprostenol use did not affect the results. In conclusion, aspirin and clopidogrel inhibited platelet aggregation, and aspirin reduced thromboxane metabolite production without affecting prostaglandin I2 metabolite synthesis. Further clinical trials of aspirin in patients with idiopathic pulmonary arterial hypertension should be performed.

Thromboxane A2/Prostaglandin H2 Receptor Activation Mediates Angiotensin II–Induced Postischemic Neovascularization

We analyzed the involvement of thromboxane (TX) A2/prostaglandin (PG) H2 (TP) receptor in ischemia-induced neovascularization in mice. Unilateral hindlimb ischemia was induced by right femoral artery ligature in male C57BL/6J mice (n=7 per group). Animals were then treated with or without TP receptor antagonist (S18886, 5 or 10 mg/kg per day; ramatroban, 10 mg/kg per day) or aspirin (30 mg/kg per day) in drinking water for 21 days. Hindlimb ischemia raised plasma level of TXB2, the stable metabolite of TXA2, by 4.7-fold. This increase was blocked by aspirin treatment whereas S18886 (5 or 10 mg/kg per day) had no effect. However, neither S 18886 nor aspirin affected postischemic neovascularization. We next assessed the putative involvement of TXA2 signaling in angiotensin II (Ang II) proangiogenic pathway. Ang II (0.3 mg/kg per day) enhanced TXB2 plasma levels by 2.6-fold over that of control (P<0.01). Ang II-induced TXB2 upregulation was reduced by cotreatment with Ang II type I receptor antagonist (candesartan, 20 mg/kg per day). Angiographic score, capillary number, and foot perfusion were improved by 1.7-, 1.7-, and 1.4-fold, respectively, in Ang II-treated mice compared with controls (P<0.05). Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content (P<0.05) and a 1.4-fold increase in the number of Mac-3-positive cells (ie, macrophages) in ischemic areas (P<0.05). Interestingly, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effects of Ang II. Endogenous activation of TXA2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. Conversely, TXA2 signaling is involved in Ang II-induced AT1-dependent vessel growth.

Do prostacyclin and thromboxane play a role in endotoxic shock?

The role of prostacyclin and thromboxane during endotoxic shock is unknown. Using new radioimmunoassay techniques, we have studied the plasma levels of stable metabolites of prostacyclin (6-keto-PGF1 alpha) and thromboxane (TxB2) in a porcine model of endotoxic shock. TxB2 levels were markedly elevated but the production of prostacyclin appears to be impaired. Correction of this prostanoid imbalance by the infusion of prostacyclin or pre-treatment with a specific thromboxane synthetase inhibitor produces significant and beneficial effects on blood pressure and pre-kallikrein activation.

Gefitinib Affects Functions of Platelets and Blood Vesselsvia Changes in Prostanoids Balance

Prostaglandins (PGs) and thromboxane (TX) produced by cyclooxygenase (COX) have a great influence on vascular systems and platelet functions. The serum levels of epidermal growth factor (EGF) and PGs were measured in patients with lung cancer treated with gefitinib, and the influence of EGF on platelet aggregation was investigated. Twenty patients were investigated. The serum level of TXB(2) increased significantly in all patients who received gefitinib for 2 weeks (before vs. after = 94.1 +/- 47.3 vs. 190.9 +/- 54.3, p<0.01). TXB(2) also increased significantly in responders without concurrent chemotherapy (before vs. after = 79.3 +/- 35.5 vs. 194.5 +/- 58.1, p<0.05), but not in non-responders (before vs. after = 106. 5 +/- 65.8 vs. 162.2 +/- 52.8, N.S.). PG 6-keto F1alpha and PGE(2) did not exhibit significant changes. Furthermore, EGF showed no significant change (after vs. before = 234 +/- 35 vs. 276 +/- 72, N.S.). Although there was no correlation between the levels of EGF and TXB(2) (N.S.), the PG 6-keto F2alpha/TXB(2) ratio decreased significantly (before vs. after = 0.054 +/- 0.018 vs 0.033 +/- 0.015, p<0.05). The secondary platelet aggregation observed after high-dose adenosine diphosphate stimulation was inhibited after a 1-minute preincubation with EGF. Platelet aggregation in patients after gefitinib administration tended to accelerate and secondary aggregation was observed after low-dose adenosine diphosphate stimulation. We conclude that careful observation is needed for patients with chronic obstructive pulmonary disease, pulmonary fibrosis, and thromboembolic diseases receiving gefitinib. Furthermore, measurement of prostanoids may be a good predictor of the beneficial and adverse effects. Moreover, the combination of gefitinib with a COX inhibitor that regulates TXA(2)/PGI(2) balance should be evaluated.

Cyclooxygenase-1 haplotype modulates platelet response to aspirin

Aspirin (acetylsalicylic acid) irreversibly inhibits platelet cyclooxygenase (COX)-1, the enzyme that converts arachidonic acid (AA) to the potent platelet agonist thromboxane (TX) A2. Despite clear benefit from aspirin in patients with cardiovascular disease (CAD), evidence of heterogeneity in the way individuals respond has given rise to the concept of 'aspirin resistance.' To evaluate the hypothesis that incomplete suppression of platelet COX as a consequence of variation in the COX-1 gene may affect aspirin response and thus contribute to aspirin resistance. Aspirin response, determined by serum TXB2 levels and AA-induced platelet aggregation, was prospectively studied in patients (n = 144) with stable CAD taking aspirin (75-300 mg). Patients were genotyped for five single nucleotide polymorphisms in COX-1 [A-842G, C22T (R8W), G128A (Q41Q), C644A (G213G) and C714A (L237M)]. Haplotype frequencies and effect of haplotype on two platelet phenotypes were estimated by maximum likelihood. The four most common haplotypes were considered separately and less common haplotypes pooled. COX-1 haplotype was significantly associated with aspirin response determined by AA-induced platelet aggregation (P = 0.004; 4 d.f.). Serum TXB2 generation was also related to genotype (P = 0.02; 4 d.f.). Genetic variability in COX-1 appears to modulate both AA-induced platelet aggregation and thromboxane generation. Heterogeneity in the way patients respond to aspirin may in part reflect variation in COX-1 genotype.

Prevention of latex sensitization in guinea pigs by a bacterial and viral filter used in anaesthesia

Preventing anaphylactic reactions as a result of natural rubber latex (NRL) proteins is an important concern in anaesthesia. The clinical relevance of a bacterial/viral filter (Pall BB25) in preventing sensitization to NRL by inhalation was tested in guinea pigs. Guinea pigs (n=8-10 in each group) were exposed to aerosolized NRL-contaminated cornstarch powder or to NRL in saline for 1 h every day for 2 weeks. The experiments were repeated with a Pall BB25 filter placed over the aerosol system. Control groups were exposed to non-contaminated cornstarch or to saline alone. Three weeks after the last exposure, specific bronchial challenge was performed and thromboxane (Tx) B2 levels in bronchoalveolar lavage fluid were measured. After bronchial challenge, the animals exposed to NRL or NRL-contaminated cornstarch with the BB25 filter in place showed a level of bronchoconstriction (i.e. the variation of pulmonary insufflation pressure) not different from controls. Conversely, those exposed to NRL or NRL-contaminated cornstarch without the filter showed a higher level of bronchoconstriction (respectively, P<0.02 and P<0.001) than control. Elevated TxB2 levels were found in the lungs of the guinea pigs, which inhaled NRL or NRL-contaminated cornstarch in the absence of a filter. Animals treated with the filter showed comparable TxB2 levels with those of control. The Pall BB25 filter efficiently protected the guinea pigs from sensitization to NRL. This filter can be used as a complementary measure for avoidance of NRL contact during surgical procedures particularly if the mechanical ventilator apparatus contain NRL devices.

Selective Cyclooxygenase-2 Inhibitors Development in Cardiovascular Medicine

At the end of September 2004, Merck & Co announced the voluntary withdrawal of rofecoxib (Vioxx) worldwide because of an increased risk of cardiovascular events. Since its approval in 1999, Vioxx, a selective cyclooxygenase-2 (COX-2) inhibitor, has been Merck & Co’s leading drug for control of acute pain and chronic pain associated with osteoarthritis, rheumatoid arthritis, and menstruation. Last year, worldwide sales of rofecoxib reached US $2.5 billion, and it is estimated that the drug was prescribed ≈10 million times per month in the United States.1 Thus, given the number of patients involved and the serious nature of the side effects, the withdrawal raised serious concerns about the safety of other selective COX-2 inhibitors, collectively called coxibs, that are on the market and those currently under development. Celecoxib (Celebrex) and rofecoxib were the first 2 coxibs approved by the US Food and Drug Administration (FDA) and launched in 1999 by Pfizer and Merck & Co, respectively. Since then, a second generation of these drugs has emerged onto the market. Valdecoxib (Bextra) was approved by the FDA and launched in 2002. In that same year, the European regulatory authority approved 2 other coxibs: etoricoxib (Arcoxia) and parecoxib sodium (Dynastat), the prodrug of valdecoxib. Today, etoricoxib and a fifth coxib, lumiracoxib (Prexige), are under consideration for FDA approval. In view of the rapid development in this area, the main concern is whether the reported cardiovascular effects of rofecoxib are a class effect applicable to all coxibs that were initially designed to reduce the gastric toxicity of nonselective COX inhibitors. Although most of the data accumulated so far would suggest a class effect related to the general mode of action of all coxibs and the physiological role of COX-2, recent in vitro data would theoretically support the hypothesis that the cardiovascular side …

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