Thromboxane Synthase Inhibitor Research Articles

Progress and challenges in lung xenotransplantation: an update.

Recent progress in genetic engineering has facilitated development of transgenic donor animals designed to overcome the known barriers to discordant xenotransplantation, and greatly accelerated progress in the field of xenotransplantation. Here we review and summarize recent progress in lung xenotransplantation, and discuss possible additional genetic modifications and other interventions that may further advance the use of pulmonary xenografts towards clinical applications based on known mechanisms of xeno lung injury. Ex-vivo lung perfusion experiments have shown that the addition of human complement (hCD46, hCD55), coagulation (hEPCR, hVWF, hTBM, hTFPI, hCD39), or anti-inflammatory pathway regulatory genes (HO-1, HLA-E), and the knockout (KO) of major porcine carbohydrates (GalT, Neu5Gc, B4Gal) have each protective effects on lung survival and function. The use of these transgenes in multitransgenic donor organs, targeting several known xenogeneic rejection mechanisms, combined with drug treatments addressing remaining known rejection pathways, have led to prolonged recipient survival of up to 31 days with in some cases preserved live-supporting organ function of the transplanted graft for several days. Pulmonary vascular resistance elevation, which has been found to be associated with high thromboxane levels and has been the major failure reason of xenogeneic lung grafts in the past years, has been successfully attenuated by the addition of a thromboxane synthase inhibitor (1-Benzylimidazole). Currently, the predominant failure mechanism of xenogeneic lung grafts is an inflammatory process, leading to vascular barrier function injury with interstitial and trachea edema. Work with other pig organs in primate models show that regimens based on costimulatory pathway blocking antibodies prolong xenograft function for months to years, suggesting that once initial lung inflammation mechanisms are fully controlled, clinically useful application of pig lung xenografts may be feasible. The use of multitransgenic donor pigs coupled with drugs targeting complement activation, coagulation, and inflammation have significantly improved the survival of xenogeneic pig lungs both during ex vivo human blood perfusion and in life-supporting in vivo models, and for the first time allowed consistent life-supporting function of lung xenografts. Overcoming delayed loss of vascular barrier function injury appears to be within reach, and will be essential to make lung xenografts a clinically relevant treatment option.

Progress in Xenogeneic Lung Transplantation Using Multi-Transgenic Donor Pigs and Targeted Supportive Drug Treatments

Purpose Significant progress in genetic engineering has enabled the generation of knock-out pigs that additionally express multiple human “transgenes” chosen to address various known xenogeneic rejection pathways. We evaluated which transgene combinations are associated with prevention of lung xenograft injury in a rigorous life-supporting pig-to-baboon lung xenograft model. Methods GTKO.hCD46 porcine donor organs with up to 4 additional genetic modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, hHO-1, HLA-E, A20 and humanized vWF were used in 48 left single lung transplants into baboons. A rationally designed “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibitor, histamine receptor blockers, and anti-GPIb Fab. Desmopressin (DDAVP) was given to the donor animals prior to lung procurement, to deplete pig endothelial VWF. Immunosuppression consisted of aCD20, ATG, MMF, aCD40, coupled with anti-IL6R moAb and/or Alpha-1 Antitrypsin. Xenograft function was assessed intermittently by transplant blood flow measurements and radiographs. Results Xenogeneic lungs with 6 genetic modifications (6-GE) transplanted into baboons survived for up to 8d (hCD55.hEPCR.hTBM.hCD39) and 9d (hEPCR.hTBM.hCD47.HO-1), vs <12hrs for most 3- or 4-GE lungs. Several other donor transgene-combinations resulted in 47d survival. Lungs of GTKO.hCD46.hCD55.hEPCR.hCD47.hTFPI and a few 3- or 4-GE phenotypes (eg including HLA-E and hvWF) consistently exhibited life-supporting (LS) lung function for 24-30h, with relatively normal macro- and microscopic lung appearance until 2-3 d. Lung xenograft failure was usually associated with rebounding anti-pig antibody titer and loss of lung vascular barrier function leading to alveolar flooding and consolidation, consistent with “delayed xenograft rejection”. Accumulated evidence suggests that, in addition to anti-non-Gal antibody, recipient NK cells (HLA-E) and donor macrophage activation (thromboxane) each play important roles to drive the residual inflammation. Conclusion Combining multi-transgenic donor organs with mechanism-directed drug treatments significantly prolongs life-supportive lung xenograft function and recipient survival. Aggregation of existing targeted genetic modifications, along with antibody depletion and mechanism-based drug additions appear likely to successfully control known pathogenic pathways, and further advance lung xenotransplantation towards clinical application. United Therapeutics SRA. NIH U19A1090959.

L‐arginine Supplementation and Thromboxane Synthase Inhibition Reverse Cerebral Ischemia in Murine Cerebral Malaria by Plasmodium berghei ANKA

Cerebral malaria (CM) is one of the most severe and lethal complications of Plasmodium falciparum infection. The mainstay treatment for CM is intravenous artesunate, nevertheless 15–20% of the patients receiving this drug still die. Vascular dysfunction, with vasoconstriction, leads to decreased cerebral blood flow, ischemia, tissue hypoxia and death in CM. Nitric oxide (NO) and arachidonic acid metabolites are important regulators of physiological cerebral blood flow through their vasodilator or vasoconstrictive properties. The objective of this study was to determine the effects of L‐arginine supplementation and pharmacological inhibition of thromboxane A2 (TXA2) synthesis on the reversal of ischemia in experimental cerebral malaria (ECM). C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) and showing clinical signs of ECM were treated subcutaneously with the NO precursor L‐arginine (50mg/kg) and/or the thromboxane synthase inhibitor ozagrel (100mg/kg). Cerebral blood flow was measured by Laser Speckle Contrast Imaging (LSCI). The adjuvant effect of L‐arginine in combination with artesunate on survival of mice with late‐stage ECM was evaluated. Animals that developed ECM showed a significant decrease (25%) in cerebral blood flow compared to non‐infected control animals. L‐arginine induced a marked increase (22.5%) in cerebral blood flow in mice with ECM one hour after administration. The administration of ozagrel, which prevents the generation of the vasoconstrictor metabolite TXA2, also led to increased (16.5%) cerebral blood flow in animals with ECM, and this was consistent with decreased levels of brain TXA2 in ozagrel‐treated compared to saline‐treated animals. The combination of ozagrel and L‐arginine showed no synergistic effect on cerebral blood flow. Treatment of animals with late‐stage ECM with artesunate plus L‐arginine increased survival compared to artesunate plus saline. Conclusion: L‐arginine and ozagrel increase cerebral blood flow, and L‐arginine increase survival in mice with cerebral malaria and show potential as adjuvant therapy for this malaria complication.Support or Funding InformationCAPES, FAPERJ, IOC‐FiocruzThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uridine Adenosine Tetraphosphate-Induced Coronary Relaxation Is Blunted in Swine With Pressure Overload: A Role for Vasoconstrictor Prostanoids

Plasma levels of the vasoactive substance uridine adenosine tetraphosphate (Up4A) are elevated in hypertensive patients and Up4A-induced vascular contraction is exacerbated in various arteries isolated from hypertensive animals, suggesting a potential role of Up4A in development of hypertension. We previously demonstrated that Up4A produced potent and partially endothelium-dependent relaxation in the porcine coronary microvasculature. Since pressure-overload is accompanied by structural abnormalities in the coronary microvasculature as well as by endothelial dysfunction, we hypothesized that pressure-overload blunts the coronary vasodilator response to Up4A, and that the involvement of purinergic receptors and endothelium-derived factors is altered. The effects of Up4A were investigated using wire-myography in isolated coronary small arteries from Sham-operated swine and swine with prolonged (8 weeks) pressure overload of the left ventricle induced by aortic banding (AoB). Expression of purinergic receptors and endothelium-derived factors was assessed in isolated coronary small arteries using real-time PCR. Up4A (10-9 to 10-5 M) failed to produce contraction in isolated coronary small arteries from either Sham or AoB swine, but produced relaxation in preconstricted arteries, which was significantly blunted in AoB compared to Sham. Blockade of purinergic P1, and P2 receptors attenuated Up4A-induced coronary relaxation more, while the effect of P2X1-blockade was similar and the effects of A2A- and P2Y1-blockade were reduced in AoB as compared to Sham. mRNA expression of neither A1, A2, A3, nor P2X1, P2X7, P2Y1, P2Y2, nor P2Y6-receptors was altered in AoB as compared to Sham, while P2Y12 expression was higher in AoB. eNOS inhibition attenuated Up4A-induced coronary relaxation in both Sham and AoB. Additional blockade of cyclooxygenase enhanced Up4A-induced coronary relaxation in AoB but not Sham swine, suggesting the involvement of vasoconstrictor prostanoids. In endothelium-denuded coronary small arteries from normal swine, thromboxane synthase (TxS) inhibition enhanced relaxation to Up4A compared to endothelium-intact arteries, to a similar extent as P2Y12 inhibition, while the combination inhibition of P2Y12 and TxS had no additional effect. In conclusion, Up4A-induced coronary relaxation is blunted in swine with AoB, which appears to be due to the production of a vasoconstrictor prostanoid, likely thromboxane A2.

Lung xenotransplantation.

This review describes the most recent progress in xeno lung transplantation (XLTx) to date. It describes the potential mechanisms of early xeno lung graft loss, as well as the latest therapeutic strategies to overcome them. Using ex-vivo perfusion models of porcine lungs with human blood, the use of genetically modified pig lungs along with novel pharmaceutical approaches has recently been studied. Strategies that have demonstrated improved lung survival include the knockout of known xenoantigens (GalTKO and N-glycolylneuraminic acid-KO), genes that regulate complement activation (hCD46 and hCD55), as well as the inflammation/coagulation cascade (human leukocyte antigen-E, human thrombomodulin, human endothelial protein C receptor, hCD47, hCD39, hCD73 and heme oxygenase-1). Furthermore, pharmacologic interventions including the depletion of pulmonary intravascular macrophages or von Willebrand factor, inhibition of thromboxane synthase and blockade of histamine receptors have also demonstrated protective effects on xeno lung grafts. Using in-vivo pig to nonhuman primate lung transplant models, these approaches have been shown to extend pulmonary xenograft survival to 5 days. The development of new multitransgenic GalTKO pigs has demonstrated prolongation of porcine xenograft survival; however, advancement in XLTx has remained frustratingly limited. Further intensive and innovative strategies including genetic manipulation of donors, as well as inflammation/coagulation dysregulation, are required to make XLTx a clinical possibility.

Involvement of thromboxane A2 in interleukin-31-induced itch-associated response in mice.

Atopic dermatitis is a chronic and severe pruritic skin disease. Interlukin-31 (IL-31) has been recently demonstrated to be one of the key pruritogens in atopic dermatitis. However, the mechanisms underlying IL-31-induced itching remains unclear. In our previous study, we have shown that thromboxane (TX) A2 is involved in itch-associated responses in mice with atopy-like skin diseases. IL-31 was given intradermally into the rostral back of ICR mice and the hind-paw scratching to the injection site were counted. Expression of TX synthase and IL-31 receptors were analyzed using immunohistochemical staining or RT-PCR in mouse skin or primary cultures of mouse keratinocytes. The concentration of TXB2, a metabolite of TXA2, in the skin and the culture medium of primary cultures of mouse keratinocytes was measured using enzyme immunoassay. The concentration of intracellular Ca2+ ions in mouse keratinocytes was measured using the calcium imaging method. An intradermal injection of IL-31 elicited scratching, an itch-related response, in mice. The scratching was inhibited by TP TXA2 receptor antagonist DCHCH. The distribution of TX synthase and IL-31RA receptor was mainly epidermal keratinocytes in the skin. The primary cultures of keratinocytes expressed the mRNAs of TX synthase and IL-31 receptors. IL-31 increased the concentration of TXB2, which was inhibited by TX synthase inhibitor sodium ozagrel and EGTA, in the skin and the culture medium of primary cultures of keratinocytes. IL-31 increased the concentration of intracellular Ca2+ ions in mouse keratinocytes. It is suggested that IL-31 elicits itch-associated responses through TXA2 produced from keratinocytes.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model.

Wild-type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody-mediated xenograft injury. One such carbohydrate is N-glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model. Livers from pigs with an α1,3-galactosyl transferase gene knockout (GalTKO) and transgenic for human membrane cofactor (hCD46) with (n=5) or without (n=7) an additional Neu5Gc gene knock out (Neu5GcKO) were perfused ex vivo with heparinized whole human blood. A drug regimen consisting of a histamine inhibitor, thromboxane synthase inhibitor, and a murine anti-human GPIb-blocking antibody fragment was given to half of the experiments in each group. Liver function tests (AST and ALT) were not significantly different between livers with and without the Neu5GcKO. GalTKO.hCD46.Neu5GcKO livers had less erythrocyte sequestration as evidenced by a higher mean hematocrit over time compared to GalTKO.hCD46 livers (P=.0003). The addition of Neu5GcKO did not ameliorate profound thrombocytopenia seen within the first 15minutes of perfusion. TXB2 was significantly less with the added drug regimen (P=.006) or the presence of Neu5GcKO (P=.017). The lack of Neu5Gc expression attenuated erythrocyte loss but did not prevent profound early onset thrombocytopenia or platelet activation, although TXB2 levels were decreased in the presence of Neu5GcKO.

Endometriosis-Derived Thromboxane A2 Induces Neurite Outgrowth.

Hyperinnervation in endometriosis is now well documented, but so far only a few neurotrophins have been identified. Since endometriotic stromal cells secrete thromboxane A2 (TXA2), we sought to determine whether TXA2, derived from endometriotic stromal cells, induces neurite outgrowth. Using primary sensory neurons derived from rat dorsal root ganglia (DRG) and ectopic endometrial stromal cells (EESCs) derived from human ovarian endometrioma tissues, we treated the primary neurons with different concentrations of U-46619, a stable TXA2 mimetic, and performed a neuronal growth assay. The primary neurons were also cocultured with a vehicle, nerve growth factor (NGF, serving as a positive control), the supernatant of EESC culture medium, or the supernatant of EESCs pretreated with ozagrel, a thromboxane synthase inhibitor, and a neuronal growth assay was performed. The total neurite length was evaluated through immunofluorescence microscopy. We found that U-46619 significantly increased the neurite outgrowth in DRG neurons in a concentration-dependent fashion ( P < .001). It also increased the number of neurite ends in a concentration-dependent fashion. Ozagrel treatment alone had no effect on the neurite growth ( P > .05), and the treatment with the supernatant of EESCs induced neurite outgrowth just as potently as that treated with NGF (positive control; P > .05). Remarkably, treatment with the EESC supernatant increased the neurite outgrowth by nearly 3-fold as compared with the control ( P < .01), but the pretreatment with ozagrel abolished the stimulatory effect of the EESC by 31.3% ( P < .05). These findings indicate that EESCs potently induce neurite outgrowth, and endometriosis-derived TXA2 is responsible, at least in part, for this neurotrophic effect.

Differential contribution of COX-1 and COX-2 derived prostanoids to cortical spreading depression—Evoked cerebral oligemia

Cortical spreading depression (CSD) is considered a significant phenomenon for human neurological conditions and one of its key signatures is the development of persistent cortical oligemia. The factors underlying this reduction in cerebral blood flow (CBF) remain incompletely understood but may involve locally elaborated vasoconstricting eicosanoids. We employed laser Doppler flowmetry in urethane-anesthetized rats, together with a local pharmacological blockade approach, to test the relative contribution of cyclooxygenase (COX)-derived prostanoids to the oligemic response following CSD. Administration of the non-selective COX inhibitor naproxen completely inhibited the oligemic response. Selective inhibition of COX-1 with SC-560 preferentially reduced the early reduction in CBF while selective COX-2 inhibition with NS-398 affected only the later response. Blocking the action of thromboxane A2 (TXA2), using the selective thromboxane synthase inhibitor ozagrel, reduced only the initial CBF decrease, while inhibition of prostaglandin F2alpha action, using the selective FP receptor antagonist AL-8810, blocked the later phase of the oligemia. Our results suggest that the long-lasting oligemia following CSD consists of at least two distinct temporal phases, mediated by preferential actions of COX-1- and COX-2-derived prostanoids: an initial phase mediated by COX-1 that involves TXA2 followed by a later phase, mediated by COX-2 and PGF2alpha.

Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor β (TGF-β) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-β signaling.

Effects of BM-573 on Endothelial Dependent Relaxation and Increased Blood Pressure at Early Stages of Atherosclerosis.

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3μM, 1 hour) and chronic (10mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

Antiplatelet pyrazolopyridines derivatives: pharmacological, biochemical and toxicological characterization

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N′-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3 h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE2 and TXB2 through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.

Possible role of thromboxane A2 in remote hind limb preconditioning-induced cardioprotection.

Remote hind limb preconditioning (RIPC) is a protective strategy in which short episodes of ischemia and reperfusion in a remote organ (hind limb) protects the target organ (heart) against sustained ischemic reperfusion injury. The present study was designed to investigate the possible role of thromboxane A2 in RIPC-induced cardioprotection in rats. Remote hind limb preconditioning was performed by four episodes of 5 min of inflation and 5 min of deflation of pressure cuff. Occlusion of the hind limb with blood pressure cuff is most feasible, non-invasive, clinically relevant, and safe method for inducing RIPC. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120-min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. The extent of myocardial infarct size along with the functional parameters including left ventricular developed pressure (LVDP), dp/dtmax, and dp/dtmin were also measured. Ozagrel (thromboxane synthase inhibitor) and seratrodast (thromboxane A2 receptor antagonist) were employed as pharmacological modulators of thromboxane A2. Remote hind limb preconditioning significantly attenuated ischemia/reperfusion-induced myocardial injury and produced cardioprotective effects. However, administration of ozagrel and seratrodast completely abolished the cardioprotective effects of RIPC suggesting the key role of thromboxane A2 in RIPC-induced cardioprotection. It may be concluded that brief episodes of preconditioning ischemia and reperfusion activates the thromboxane synthase enzyme that produces thromboxane A2, which may elicit cardioprotection either involving humoral or neurogenic pathway.

Abstract 600: Novel Assays to Determine in vivo Platelet Activation

In vivo platelet activation is associated with several pathologic entities, including acute coronary syndrome, essential thrombocythemia, anti-phospholipid antibody syndrome, diabetes and metabolic syndrome. The need for more robust biomarkers with which to assess platelet activation in vivo in human diseases is well recognized. Our lab has developed two novel assays to study in vivo platelet activation and rate of turnover. During platelet activation, malondialdehyde (MDA) is produced by the thromboxane synthase in amounts equimolar to thromboxane A2 and also non-enzymatically by lipid peroxidation resulting from the oxidative processes that accompany activation. MDA is a reactive dicarbonyl that reacts with amines, notably lysines on proteins, yielding covalent modifications of the proteins that then accumulate over the lifetime of the platelet. We developed an LC/MS/MS method for quantification of the most stable of three MDA adducts, the dilysyl-MDA crosslink, employing a [13C12] labeled internal standard. We found that activation of platelets with arachidonic acid leads to an increase in the levels of dilysyl-MDA crosslinks in platelets, which is inhibited by the thromboxane synthase inhibitor, ozagrel, by the cyclooxygenase inhibitor, aspirin, and by scavengers of reactive carbonyls, 3- methoxysalicylamine and Salicylamine. High platelet turnover has been associated with increased risk for thrombosis and failure of antiplatelet agents. We propose a novel approach to studying platelet turnover by labeling platelets in vivo by oral administration of aspirin containing a deuterium labeled acetyl group (d3-aspirin). We can measure the clearance of d3-labeled platelets by using LC/MS/MS to measure the tryptic peptide (SLK) of COX-1 labeled with d3. This approach avoids exposure to radioactivity and the artifact resulting from manipulation of platelets labeled ex vivo. The results of this study provide two novel assays with the potential to serve as markers of in vivo platelet activation and turnover, which may be useful in predicting thrombotic risk and efficacy of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity and high rate of platelet turnover.

Mechanisms underlying uridine adenosine tetraphosphate-induced vascular contraction in mouse aorta: Role of thromboxane and purinergic receptors

Uridine adenosine tetraphosphate (Up4A), a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorders and induces aortic contraction through activation of cyclooxygenases (COXs). We and others demonstrated that activation of A1 or A3 adenosine receptors (ARs) results in vascular contraction via thromboxane (TX) A2 production. However, the mechanisms of Up4A-induced vascular contraction in mouse aorta are not understood. We hypothesize that Up4A-induced aortic contraction is through COX-derived TXA2 production, which requires activation of A1 and/or A3AR. Concentration responses to Up4A were conducted in isolated aorta. The TXB2 production, a metabolite of TXA2, was also measured. Up4A (10−9-10−5M) produced a concentration-dependent contraction >70%, which was markedly attenuated by COX and COX1 but not by COX2 inhibition. Notably, Up4A-induced aortic contraction was blunted by both TX synthase inhibitor ozagrel and TXA2 receptor (TP) antagonist SQ29548. Surprisingly, A3AR deletion had no effect on Up4A-induced contraction. Moreover, A1AR deletion or antagonism as well as A1/A3AR deletion potentiated Up4A-induced aortic contraction, suggesting a vasodilator influence of A1AR. In contrast, non-selective purinergic P2 receptor antagonist PPADS significantly blunted Up4A-induced aortic contraction to a similar extent as selective P2X1R antagonist MRS2159, the latter of which was further reduced by addition of ozagrel. Endothelial denudation almost fully attenuated Up4A-induced contraction. Furthermore, Up4A (3μM) increased TXB2 formation, which was inhibited by either MRS2159 or ozagrel. In conclusion, Up4A-induced aortic contraction depends on activation of TX synthase and TP, which partially requires the activation of P2X1R but not A1 or A3AR through an endothelium-dependent mechanism.

Role of the non-enzymatic metabolite of eicosapentaenoic acid, 5-epi-5-F3t-isoprostane in the regulation of [ (3)H]D-aspartate release in isolated bovine retina.

We have evidence that F2-isoprostanes (F2-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F3-IsoPs are generated in mammals, in vivo, their pharmacological actions on neurotransmitter release remain unknown. In this study, we investigated the effect of 5-epi-5-F3t-IsoP on K(+)-evoked [(3)H]D-aspartate release in isolated bovine retina using the superfusion method. Furthermore, we examined the role of arachidonic acid metabolites in the regulation of the neurotransmitter release by this novel IsoP. In the concentration range, 0.01nM-0.1µM, 5-epi-5-F3t-IsoP inhibited K(+)-evoked [(3)H]D-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9% at 0.1µM (IC30=1nM). The prostanoid receptor antagonists, AH 6809 (EP1-3/DP; 10µM), SC 51322 (EP1; 10µM) and SC 19220 (EP1; 1µM) partially reversed 5-epi-5-F3t-IsoP-mediated inhibition of K(+)-induced [(3)H]D-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3μM) unmasked a biphasic action of 5-epi-5-F3t-IsoP that was inhibitory at lower (0.1-10pM) and stimulatory at higher concentrations (≥0.1nM). The prostanoid pathway antagonists, BAY-u3405 (10μM; TP/DP-receptors), SQ 29548 (10μM; TP-receptor) and ozagrel (10μM; Tx-synthase inhibitor) abolished the stimulatory action of the 5-epi-5-F3t-IsoP (0.1μM) on neurotransmitter release. In conclusion, 5-epi-5-F3t-IsoP attenuates K(+)-induced [(3)H]D-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP1-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5-epi-5-F3t-IsoP that is inhibitory at low concentrations and stimulatory at higher concentrations. Products of the thromboxane synthase pathway may partially account for the stimulatory action of this F3-IsoP on isolated bovine retina.

5-Substituted pyridylisoxazoles as effective inhibitors of platelet aggregation

A series of 5-substituted 3-pyridylisoxazoles were synthesized using [3+2] cycloaddition of nitrile oxides to alkynes with variation of substituents at position 5 of the isoxazole ring without additional synthetic stages and with retention of 2-pyridyl-, 3-pyridyl, and 4-pyridyl substituents at position 3 of the isoxazole ring. Substituted pyridylisoxazoles are the potential antiaggregatory agents showing in vitro activity in the concentration range from 1•10−6 mol L−1 to 1•10−4 mol L−1 toward the human platelet rich blood plasma with arachidonic acid being used as the inductor of aggregation. These compounds do not act as cyclooxygenase or thromboxane synthase inhibitors, nor as thrombin inhibitors.

Combined Thromboxane Synthase Inhibition and H2-Receptor Blockade Prevents PVR Elevation During Xenogenic Pig Lung Perfusion.

Purpose: Xenogenic lung perfusion studies revealed that inflammation and activation of platelets and coagulation cascade are important factors of xenogenic rejection mechanisms, contributing to increased pulmonary vascular resistance (PVR) and organ injury. Here we evaluate effects of combined thromboxane synthase inhibitor and histamine blocker administration on pig lung function and phenomena associated with “hyperacute lung rejection” (HALR). Methods: 6 GalTKO.hCD46.hCD55 pig lungs were perfused for 4h with fresh heparinized human blood: one lung side was “treated” with a single dose of Famotidine and 1-Benzylimidazole (11.2 and 112mg/L of blood, respectively) to the perfusate, whereas the contralateral non-treated side served as “control”. Results: All organs kept adequate blood oxygenation and “survived” until elective termination. Controls showed a PVR rise within the first hour (mean PVR at 30' 286±83 mmHg*min/L) whereas treated lungs exhibited no PVR rise throughout the experiment (33±3 at 30'; p<0.01). Terminal tissue wet/dry-ratio was significantly lower in treated lungs compared to control experiments (3.9±0.15, vs. 5.0±0.16; p<0.01). Platelet and neutrophil sequestration were not altered by the treatment. Analysis of platelet, complement and coagulation pathway activation, thromboxane, and histamine levels are in progress. Conclusion: Combined histamine and thromboxane blockade prevents PVR elevation and inhibits loss of vascular barrier function when GalTKO.hCD46.hCD55 lungs are perfused with human blood, despite platelet sequestration. The finding demonstrates that innate immune pathways are not fully inhibited by this gene construct, and are pivotal mediators of HALR despite efficient complement activation regulation, as previously reported for the GalTKO.hCD46 construct. While the relative importance of these pathways remains untested, and mechanistic correlates require further elucidation, this observation will likely be readily translatable into preclinical in vivo xenogenic lung transplant models. DISCLOSURES:Ayares, D.: Employee, Revivicor.

(698) - Combined Thromboxane Synthase Inhibition and H2-Receptor Blockade Prevents PVR Elevation During GalTKO.hCD46.hCD55 Pig Lung Perfusion With Human Blood

Recent xenogenic lung perfusion studies revealed that inflammation and activation of platelets and coagulation cascade are important factors of xenogenic rejection mechanisms, contributing to increased pulmonary vascular resistance (PVR) and organ injury. Here we evaluate effects of combined thromboxane synthase inhibitor and histamine blocker administration on pig lung function and phenomena associated with

Lysophosphatidic Acid Induces Shear Stress–dependent Contraction in Mouse Aortic Strip In Situ

We previously reported that lysophosphatidic acid (LPA) regulates Ca²⁺ influx of fluid flow in stimulated endothelial cells and that LPA and shear stress showed increment and suppressive effects on phenylephrine-induced vasoconstriction and acetylcholine-induced vasodilatation, respectively. However, a vasoconstrictive effect of LPA alone in the presence of shear stress was not found. The present study examined the effect of LPA alone in the presence of shear stress on Ca²⁺ responses in endothelial and smooth muscle cells and contraction in mouse aortic strip using real-time 2-photon laser scanning microscopy and a custom-made parallel-plate flow chamber. Application of micromolar LPA and high shear stress elicited movement of endothelial cells after Ca²⁺ responses. The endothelial cells moved along the major axis of smooth muscle cells, a direction that was identical to that found during vasoconstriction evoked by the application of phenylephrine. The frequency of Ca²⁺ oscillations in smooth muscle cells was highest according to endothelial movement. Vasoconstriction evoked by LPA and shear stress was significantly reduced by the application of a thromboxane A₂ receptor antagonist, a cyclooxygenase inhibitor, and a thromboxane synthase inhibitor. These results suggest that micromolar LPA and high shear stress elicit vasoconstriction that is caused by Ca²⁺-dependent contraction in medial smooth muscle cells. Thromboxane A₂ may be involved in that response.