Release Of Thromboxane A2 Research Articles

Targeting Rap1b signaling cascades with CDNF: Mitigating platelet activation, plasma oxylipins and reperfusion injury in stroke

Cerebral reperfusion injury in stroke, stemming from interconnected thrombotic and inflammatory signatures, often involves platelet activation, aggregation and its interaction with various immune cells, contributing to microvascular dysfunction. However, the regulatory mechanisms behind this platelet activation and the resulting inflammation are not well understood, complicating the development of effective stroke therapies. Utilizing animal models and platelets from hemorrhagic stroke patients, our research demonstrates that human cerebral dopamine neurotrophic factor (CDNF) acts as an endogenous antagonist, mitigating platelet aggregation and associated neuroinflammation. CDNF moderates mitochondrial membrane potential, reactive oxygen species production, and intracellular calcium in activated platelets by interfering with GTP binding to Rap1b, thereby reducing Rap1b activation and downregulating the Rap1b-MAPK-PLA2 signaling pathway, which decreases release of the pro-inflammatory mediator thromboxane A2. Additionally, CDNF reduces the inflammatory response in BV2 microglial cells co-cultured with activated platelets. Consistent with ex vivo findings, subcutaneous administration of CDNF in a rat model of ischemic stroke significantly reduces platelet activation, aggregation, lipid mediator production, infarct volume, and neurological deficits. In summary, our study highlights CDNF as a promising therapeutic target for mitigating platelet-induced inflammation and enhancing recovery in stroke. Harnessing the CDNF pathway may offer a novel therapeutic strategy for stroke intervention.

LPA-Induced Thromboxane A2-Mediated Vasoconstriction Is Limited to Poly-Unsaturated Molecular Species in Mouse Aortas.

We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.

Comirnaty-induced cardiopulmonary distress and other symptoms of complement-mediated pseudo-anaphylaxis in a hyperimmune pig model: Causal role of anti-PEG antibodies

BackgroundComirnaty, Pfizer-BioNTech's polyethylene-glycol (PEG)-containing Covid-19 vaccine, can cause hypersensitivity reactions (HSRs), or rarely, life-threatening anaphylaxis in a small fraction of immunized people. A causal role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet proven in an animal model. The aim of this study was to provide such evidence using pigs immunized against PEG, which displayed very high levels of anti-PEG antibodies (Abs). We also aimed to find evidence for a role of complement activation and thromboxane A2 release in blood to explore the mechanism of anaphylaxis. MethodsPigs (n = 6) were immunized with 0.1 mg/kg PEGylated liposome (Doxebo) i.v., and the rise of anti-PEG IgG and IgM were measured in serial blood samples with ELISA. After ∼2–3 weeks the animals were injected i.v. with 1/3 human dose of the PEGylated mRNA vaccine, Comirnaty, and the hemodynamic (PAP, SAP) cardiopulmonary (HR, EtCO2,), hematological (WBC, granulocyte, lymphocyte and platelet counts) parameters and blood immune mediators (anti-PEG IgM and IgG antibodies, thromboxane B2, C3a) were measured as endpoints of HSRs (anaphylaxis). ResultsThe level of anti-PEG IgM and IgG rose 5–10-thousand-fold in all of 6 pigs immunized with Doxebo by day 6, after which time all animals developed anaphylactic shock to i.v. injection of 1/3 human dose of Comirnaty. The reaction, starting within 1 min involved maximal pulmonary hypertension and decreased systemic pulse pressure amplitude, tachycardia, granulo- and thrombocytopenia, and skin reactions (flushing or rash). These physiological changes or their absence were paralleled by C3a and TXB2 rises in blood. ConclusionsConsistent with previous studies, these data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty, which involves complement activation, and, hence, it represents C activation-related pseudo-anaphylaxis. The setup provides the first large-animal model for mRNA-vaccine-induced anaphylaxis in humans.

The COVID-19 mRNA vaccine Comirnaty induces anaphylactic shock in an anti-PEG hyperimmune large animal model

Abstract Background The polyethylene-glycol (PEG)-containing COVID-19 mRNA vaccines can cause hypersensitivity reactions (HSRs), or rarely, life-threatening anaphylaxis in a small fraction of patients. The role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet been proven in an animal model. Purpose The aim of this study was to provide such evidence using an anti-PEG hyperimmune porcine model wherein the Ab levels are increased by PEGylated liposomes called Doxebo. We also aimed to find evidence for a role of complement activation and thromboxane A2 release in blood. Methods Experimental pigs (n=6) were immunized with 0.1 mg/kg BW Doxebo i.v., and the rise of anti-PEG Abs were measured with ELISA. 2-3 weeks later the animals were injected with 1/3 human dose of Pfizer’s COVID-19 mRNA vaccine Comirnaty i.v., and the hemodynamic, hematological and blood immune mediator changes were measured to assess the severity of hypersensitivity reaction (HSR). The endpoints were PAP, SAP, HR, EtCO2, WBC, granulocyte, lymphocyte and platelet counts, C3a and plasma TXB2. Results The level of anti-PEG IgM rose 5-10-thousand-fold in all pigs immunized with Doxebo by day 10. Each of the 6 animals developed an anaphylactic shock to injection of 1/3 human dose of Comirnaty that required cardiopulmonary resuscitation in 4 cases. The reaction, starting within 1 min involved maximal pulmonary hypertension (PAP 13±2 before vs. 37±6mmHg after Comirnaty, median±MAD, P<0.05) and severe systemic hypotension (SAP 63±8 before vs. 42±11mmHg after Comirnaty, median±MAD, P<0.05), tachycardia, granulo- and thrombocytopenia, and skin flushing or rash. These hemodynamic changes were paralleled by C3a and TXB2 levels in blood. Conclusion We have established the first relevant large animal model of anaphylactic reaction induced by an mRNA vaccine. Consistent with previous studies, our current data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty, which involves complement activation, and, hence, it represents CARPA (complement activation-related pseudo-allergy).

Abstract 317: Development of a Swine Model of Rapidly-Induced Septic Shock Using Single Lipopolysaccharide Infusion and Titratable Inhaled Nitric Oxide to Prevent Severe Pulmonary Vasoconstriction

Objective: A serious limitation of LPS-induced swine models of septic shock is severe pulmonary vasoconstriction triggered, in part, by the pulmonary release of thromboxane A2. Efforts to overcome this limitation have included a stepwise increase in LPS dosing, intravenous aspirin derivatives, and inhaled nitric oxide (NO). We studied the effects of titrated inhaled NO given coincident with rapid LPS-infusion aimed at inducing the key features of septic shock over a shorter time period. Methods: We developed a LabVIEW-controlled system for inhaled NO delivery and monitoring. Exploratory studies showed that an NO concentration of up to 300 ppm sufficed to prevent significant pulmonary vasoconstriction despite delivery of a full LPS dose (12.6 μg/kg) in 30 minutes. We used this approach in 6 male domestic pigs (39.2 - 41.1 kg) and assessed hemodynamic and metabolic effects over 6 hours. NO and LPS infusion were started concurrently. Fluid resuscitation and vasopressor therapy were guided by macrohemodynamic measurements using custom-developed LabVIEW-controlled closed-loop algorithms. Results: Shown are medians with IQ25 and IQ75. LPS infusion increased pulmonary vascular resistance from 112 [72, 127] at baseline to 213 [120, 313] dynes-sec/cm 5 after 20 minutes. NO (139 [131, 166] ml/min) rapidly blunted the effect preventing the mean pulmonary artery pressure from exceeding 30 mmHg, avoiding detrimental effects on right ventricular function throughout the experiment. NO was rapidly de-escalated over 30 minutes using a closed-loop algorithm developed to prevent the mean pulmonary artery pressure from exceeding 30 mmHg. Within 60 minutes of LPS infusion start, animals began to develop key features of septic shock including tachycardia, hypotension, and systemic vasodilation triggering fluid resuscitation and norepinephrine administration. Additionally, blood lactate increased from 1.06 [0.90, 1.45] at baseline to 4.14 [2.43, 8.78] mmol/l after 3 hours remaining stable for an additional hour and subsequently decreasing in 3 and increasing in the other 3 animals, with half of the animals surviving the 6-h period. Conclusion: We developed a swine model of rapidly-induced key features of septic shock useful to explore early septic shock management.

Thrombo-Inflammation in COVID-19 and Sickle Cell Disease: Two Faces of the Same Coin.

People with sickle cell disease (SCD) are at greater risk of severe illness and death from respiratory infections, including COVID-19, than people without SCD (Centers for Disease Control and Prevention, USA). Vaso-occlusive crises (VOC) in SCD and severe SARS-CoV-2 infection are both characterized by thrombo-inflammation mediated by endothelial injury, complement activation, inflammatory lipid storm, platelet activation, platelet-leukocyte adhesion, and activation of the coagulation cascade. Notably, lipid mediators, including thromboxane A2, significantly increase in severe COVID-19 and SCD. In addition, the release of thromboxane A2 from endothelial cells and macrophages stimulates platelets to release microvesicles, which are harbingers of multicellular adhesion and thrombo-inflammation. Currently, there are limited therapeutic strategies targeting platelet-neutrophil activation and thrombo-inflammation in either SCD or COVID-19 during acute crisis. However, due to many similarities between the pathobiology of thrombo-inflammation in SCD and COVID-19, therapies targeting one disease may likely be effective in the other. Therefore, the preclinical and clinical research spurred by the COVID-19 pandemic, including clinical trials of anti-thrombotic agents, are potentially applicable to VOC. Here, we first outline the parallels between SCD and COVID-19; second, review the role of lipid mediators in the pathogenesis of these diseases; and lastly, examine the therapeutic targets and potential treatments for the two diseases.

Raynaud's Phenomenon: A Brush Up!

Raynaud's Phenomenon: A Brush Up!

A naturally hypersensitive porcine model may help understand the mechanism of COVID-19 mRNA vaccine-induced rare (pseudo) allergic reactions: complement activation as a possible contributing factor

A tiny fraction of people immunized with lipid nanoparticle (LNP)-enclosed mRNA (LNP-mRNA) vaccines develop allergic symptoms following their first or subsequent vaccinations, including anaphylaxis. These reactions resemble complement (C) activation-related pseudoallergy (CARPA) to i.v. administered liposomes, for which pigs provide a naturally oversensitive model. Using this model, we injected i.v. the human vaccination dose (HVD) of BNT162b2 (Comirnaty, CMT) or its 2-fold (2x) or 5-fold (5x) amounts and measured the hemodynamic changes and other parameters of CARPA. We observed in 6 of 14 pigs transient pulmonary hypertension along with thromboxane A2 release into the blood and other hemodynamic and blood cell changes, including hypertension, granulocytosis, lymphopenia, and thrombocytopenia. One pig injected with 5x CMT developed an anaphylactic shock requiring resuscitation, while a repeat dose failed to induce the reaction, implying tachyphylaxis. These typical CARPA symptoms could not be linked to animal age, sex, prior immune stimulation with zymosan, immunization of animals with Comirnaty i.v., or i.m. 2 weeks before the vaccine challenge, and anti-PEG IgM levels in Comirnaty-immunized pigs. Nevertheless, IgM binding to the whole vaccine, used as antigen in an ELISA, was significantly higher in reactive animals compared to non-reactive ones. Incubation of Comirnaty with pig serum in vitro showed significant elevations of C3a anaphylatoxin and sC5b-9, the C-terminal complex. These data raise the possibility that C activation plays a causal or contributing role in the rare HSRs to Comirnaty and other vaccines with similar side effects. Further studies are needed to uncover the factors controlling these vaccine reactions in pigs and to understand their translational value to humans.

English

BACKGROUND Platelet size, measured as mean platelet volume (MPV), is a marker of platelet function and is positively associated with indicators of platelet activity, including aggregation and release of thromboxane A2, platelet factor 4, and thromboglobulin.1 Larger platelets are metabolically more active, produce more prothrombotic factors, aggregate more easily & act as index of homeostasis and its dysfunction thrombosis.2 The purpose of this study was to examine the relationship between platelet indices and stroke, as well as its severity and outcome. METHODS This was a prospective observational case control study. This study was conducted with 105 non-diabetic, non-hypertensive ischemic stroke patients who had no history of previous thrombotic events and who had not previously taken any antiplatelet medications. These patients were examined within 24 hours of onset of symptoms and severity of stroke was calculated by Canadian neurological scale (CNS). The results were compared with 105 age and sex match controls. RESULTS Mean age of patients was 61.72 ± 12 and of controls was 62.85 ± 10.68. Based on the CNS score, participants were allocated into two groups; the first group were those who had a comprehension deficit (1st group, 43 patients) and the second group were those without a comprehension deficit (2nd group, 62 patients). Mean values for platelet distribution width (PDW) & MPV in 1st group was 18.329 and 12.55 respectively and in 2nd group was 16.98 and 11.48 respectively. The mean value of PDW and MPV for stroke patients was 17.53 ± 0.76 and 11.92 ± 0.58 and was significantly higher than mean value of PDW & MPV respectively in controls, which were 15.47 ± 0.26 and 10.43 ± 0.23. PDW & MPV was found to be significantly associated with severity of motor deficit. CONCLUSIONS Larger studies may be required to determine its utility in day-to-day clinical practice. However, platelet indices can be used for predicting the severity of deficit in patients of acute ischemic stroke. KEYWORDS Platelet Indices, Stroke

Intravital Imaging Identifies the VEGF-TXA2 Axis as a Critical Promoter of PGE2 Secretion from Tumor Cells and Immune Evasion.

Prostaglandin E2 (PGE2) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE2, little is known whether PGE2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A2 (TXA2) triggers Ca2+ transients in tumor cells, culminating in PGE2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca2+ transients were monitored as the surrogate marker of PGE2 secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca2+ transients is markedly higher in vivo than in vitro. The TXA2 receptor was indispensable for the Ca2+ transients in vivo, high intratumoral PGE2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca2+ transients and reduced TXA2 and PGE2 concentrations in tumor tissues. These results identify the VEGF-TXA2 axis as a critical promoter of PGE2-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies. SIGNIFICANCE: This study identifies the VEGF-TXA2 axis as a potentially targetable regulator of PGE2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies.

Drupin, a thrombin-like protease prompts platelet activation and aggregation through protease-activated receptors.

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from theFicus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases andthe nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2 leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Thromboxane A2 Synthase and Thromboxane Receptor Deletion Reduces Ischaemia/Reperfusion-Evoked Inflammation, Apoptosis, Autophagy and Pyroptosis.

Enhancement of thromboxane A2 (TXA2) synthase (TXAS) activity, TXA2 release, and thromboxane prostanoid (TP) receptor activation leads to vasoconstriction and oxidative injury. We explored whether genetic deletion of TXAS/TXA2/TP signalling may reduce renal ischaemia/reperfusion (I/R) injury in mice. Renal haemodynamics and function were evaluated in TXAS+/+TP+/+ (wild-type, WT), TXAS-/- (TXS-/-), TP-/- and TXAS-/-TP-/- (double knockout, dKO) mice in response to intravenous TXA2 mimetic-U46619 and 45-minute renal ischaemia and 4-hour reperfusion injury. We examined renal TXAS and TP expression, blood urea nitrogen (BUN) and creatinine, reactive oxygen species (ROS) amount, pro-inflammatory cytokines and pathophysiologic mechanisms, including apoptosis, autophagy and pyroptosis under I/R injury. Renal I/R enhanced the levels of TXAS, TP, nuclear factor-κB, nicotinamide adenine dinucleotide phosphate oxidase gp91, Bax/Bcl-2/caspase-3/apoptosis, Beclin-1/LC3-II/autophagy, caspase-1/gasdermin D/interleukin-1β/pyroptosis, renal thromboxane B2 (TXB2) concentration, ROS amount, plasma BUN, creatinine and TXB2 and decreased renal endothelial nitric oxide synthase expression in WT mice. All these enhanced parameters were significantly decreased in three KO mice. Intravenous U46619 significantly decreased renal microcirculation and enhanced gp91 and Bax/Bcl-2 in WT and TXS-/- but not TP-/- in dKO mice. I/R significantly decreased renal microcirculation in all mice; however, the time for recovery to baseline renal blood flow level was significantly shortened in TXS-/-, TP-/-and dKO mice versus WT mice. Blockade of TXAS/TP signalling attenuated I/R-enhanced pro-inflammatory cytokine profile. Blockade of TXAS/TXA2/TP signalling confers renal protection against I/R injury through the actions of anti-oxidation, anti-inflammation, anti-apoptosis, anti-autophagy and anti-pyroptosis.

Stability of thromboxane in blood samples.

Introduction: Conventional venous blood collection requires a puncture with a needle through the endothelium of a vessel. The endothelial injury causes activation of circulating platelets and the release of thromboxane A2. The aim of the study was to investigate if platelets continue to form thromboxane A2 in the blood tube after sample collection, but such synthesis would give false information about the actual circulating thromboxane A2 value.Methods: Thromboxane B2 is a biologically inactive but stable metabolite of thromboxane A2 and can be measured in blood samples by a standard enzyme immunoassay. Thromboxane B2 measurements reflect thromboxane A2 concentration. Blood samples were collected in 3.2% sodium citrate vials and EDTA vials from ten individuals and centrifuged and frozen at different time points (0, 30, and 120 minutes). Plasma aliquots were transferred to and frozen in 1.8 mL polypropylene tubes and the citrate samples were also transferred to and frozen in propylene tubes containing indomethacin.Results: Concentrations of thromboxane B2 in plasma samples collected in citrate vials and stored in propylene tubes increased very rapidly as the samples were left for longer after sampling and allowed to stand at room temperature. After 120 minutes, the amount of thromboxane B2 was 400% higher than in the reference sample at time zero. In comparison, thromboxane B2 concentration was about 200% higher in the 120-minute samples compared to the reference in samples collected in citrate vials but stored in indomethacin tubes. In samples collected in EDTA vials, a 10% reduction in thromboxane B2 concentration in the 120-minute samples was observed.Conclusion: Storage conditions, type of sampling vial and time from sampling until sample processing (centrifuging) has a major impact on thromboxane B2 stability.

The role of coagulation and platelets in colon cancer-associated thrombosis.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Leukotriene E4 induces airflow obstruction and mast cell activation through the cysteinyl leukotriene type 1 receptor

Leukotriene (LT) E4 is the final active metabolite among the cysteinyl leukotrienes (CysLTs). Animal studies have identified a distinct LTE4 receptor, suggesting that current cysteinyl leukotriene type 1 (CysLT1) receptor antagonists can provide incomplete inhibition of CysLT responses. We tested this hypothesis by assessing the influence of the CysLT1 antagonist montelukast on responses induced by means of inhalation of LTE4 in asthmatic patients. Fourteen patients with mild intermittent asthma and 2 patients with aspirin-exacerbated respiratory disease received 20mg of montelukast twice daily and placebo for 5 to 7days in a randomized, double-blind, crossover study (NCT01841164). The PD20 value was determined at the end of each treatment period based on an increasing dose challenge. Measurements included lipid mediators in urine and sputum cells 4hours after LTE4 challenge. Montelukast completely blocked LTE4-induced bronchoconstriction. Despite tolerating an at least 10 times higher dose of LTE4 after montelukast, there was no difference in the percentage of eosinophils in sputum. Urinary excretion of all major lipid mediators increased after LTE4 inhalation. Montelukast blocked release of the mast cell product prostaglandin (PG) D2, as well as release of PGF2α and thromboxane (Tx) A2, but not increased excretion of PGE2 and its metabolites or isoprostanes. LTE4 induces airflow obstruction and mast cell activation through the CysLT1 receptor.

Diabetes modifies the role of prostanoids and potassium channels which regulate the hypereactivity of the rabbit renal artery to BNP.

Diabetic nephropathy is associated with increased risk of cardiovascular disease. B-type natriuretic peptide (BNP) plays an important role in cardiovascular pathophysiology and therapeutics. The aim of the present study was to investigate the influence of experimental diabetes on the mechanisms that regulate the relaxant response of the rabbit renal artery to BNP. Arterial relaxations to BNP were enhanced in diabetic rabbits. Indomethacin enhanced BNP-induced relaxation in control rabbits but showed no effect in diabetic rabbits. BNP-induced release of thromboxane A2 or prostacyclin was not different in both groups of animals. Iberiotoxin had no effect on relaxations to BNP in both groups of animals. Charybdotoxin displaced to the right the concentration-response curve to BNP in both group of animals, and inhibited BNP-induced relaxation only in diabetic rabbits. Glibenclamide did not modify the BNP-induced relaxations in control rabbits, but inhibited it in diabetic rabbits. These results suggest that diabetes induces hypereactivity of the rabbit renal artery to BNP by mechanisms that at least include (1) a reduced vasoconstrictor influence of arachidonic acid metabolites via cyclooxygenase 2, which is not related with changes in thromboxane A2 and prostacyclin release from the arterial wall and (2) a selectively increased modulatory activity of KATP and endothelial IKCa channels.

Aqueous Extract of Tournefortia sarmentosa Stem Inhibits ADP-induced Platelet Aggregation

Tournefortia sarmentosa is a Chinese herbal medicine used as an antioxidant, detoxicant, and anti-inflammatory agent. The present study evaluated the effect of T. sarmentosa on adenosine diphosphate-induced platelet aggregation. Our results showed that aqueous stem extract of T. sarmentosa inhibited adenosine diphosphate-induced platelet aggregation. In addition, we found components in T. sarmentosa, including caffeic acid, rosmarinic acid, salvianolic acid, play important roles in mediating adenosine diphosphate-induced platelet aggregation suppression. The stem extract of T. sarmentosa inhibited the adenosine diphosphate receptor P2Y12-mediated cyclic AMP production. Caffeic acid inhibited P2Y1-induced calcium influx. Furthermore, treatment of platelets with T. sarmentosa, or the components in stem extract of T. sarmentosa, suppressed adenosine diphosphate-induced release of thromboxane A2 and arachidonic acid and surface PAC-1 expression. These data demonstrate the aqueous stem extract of T. sarmentosa significantly suppressed platelet aggregation through P2Y1 and P2Y12 receptor signal pathways. The antiaggregation properties found in the stem extract of T. sarmentosa might help to prevent cardiovascular disease.

Hyperglycaemia in pregnant rats causes sex-related vascular dysfunction in adult offspring: role of cyclooxygenase-2.

What is the central question of this study? Hyperglycaemia during pregnancy induces vascular dysfunction and hypertension in male offspring. Given that female offspring from other fetal programming models are protected from the effects of fetal insult, the present study investigated whether there are sex differences in blood pressure and vascular function in hyperglycaemia-programmed offspring. What is the main finding and its importance? We demonstrated that hyperglycaemia in pregnant rats induced vascular dysfunction and hypertension only in male offspring. We found sex differences in oxidative stress and cyclooxygenase-2-derived prostanoid production that might underlie the vascular dysfunction. These differences, particularly in resistance arteries, may in part explain the absence of hypertension in female offspring born to hyperglycaemic dams. Exposure to maternal hyperglycaemia induces hypertension and vascular dysfunction in adult male offspring. Given that female offspring from several fetal programming models are protected from the effects of fetal insult, in this study we analysed possible differences relative to sex in blood pressure and vascular function in hyperglycaemia-programmed offspring. Hyperglycaemia was induced on day 7 of gestation (streptozotocin, 50mgkg-1 ). Blood pressure, acetylcholine and phenylephrine or noradrenaline responses were analysed in the aorta and mesenteric resistance arteries of 3-, 6- and 12-month-old male and female offspring. ThromboxaneA2 release was analysed with commercial kits and superoxide anion (O2- ) production by dihydroethidium-emitted fluorescence. Male but not female offspring of hyperglycaemic dams (O-DR) had higher blood pressure than control animals (O-CR). Contraction in response to phenylephrine increased and relaxation in response to acetylcholine decreased only in the aorta from 12-month-old male O-DR and not in age-matched O-CR. Contractile and vasodilator responses were preserved in both the aorta and mesenteric resistance arteries from female O-DR of all ages. Pre-incubation with tempol, superoxide dismutase, indomethacin, NS-398, furegrelate or SQ29548 decreased contraction in response to phenylephrine and potentiated relaxation in response to acetylcholine in 12-month-old male O-DR aorta. In this artery, thromboxaneA2 release and O2- generation were greater in O-DR than O-CR groups. In conclusion, exposure to hyperglycaemia in utero results in sex-specific and age-dependent hypertension. The fact that vascular function is preserved in female O-DR may in part explain the absence of hypertension in this group. In contrast, the peripheral artery dysfunction associated with increased cyclooxygenase-2-derived production of vasoconstrictor prostanoids could underlie the increased blood pressure in male O-DR.

Salvia miltiorrhiza Bunge (Danshen) extract attenuates permanent cerebral ischemia through inhibiting platelet activation in rats

Ethnopharmacological relevanceDanshen is a crude herbal drug isolated from dried roots of Salvia miltiorrhiza Bunge. This plant is widely used in oriental medicine for the treatment of cardiovascular and cerebrovascular diseases. The supercritical CO2 extract from Danshen (SCED) (57.85%, 5.67% and 4.55% for tanshinone IIA, tanshinone I and cryptotanshinone respectively) was studied in this article, whose potential molecular mechanism remains unclear, especially in anti-thrombosis. Aim of the studyThe present study was designed to observe the protective effect of SCED on ischemic stroke in rats and to explore the underlying anti-thrombosis mechanism. Materials and methodsFollowing induction of cerebral ischemia in rats by permanent middle cerebral artery occlusion (pMCAO). Neurological defect score, cerebral blood flow, infarct size, and brain edema were measured to evaluate the injury. Arteriovenous shunt thrombosis model and adenosine 5′-diphosphate (ADP) induced acute pulmonary embolism model were conducted to estimate the antithrombotic effect of SCED. In order to investigate the effects of SCED on platelet aggregation, rat platelet-rich-plasma (PRP) were incubated with SCED prior to the addition of the stimuli (ADP or 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619)). Aggregation was monitored in a light transmission aggregometer. Inhibitory effect of SCED on thromboxane A2 (TXA2) release was detected by ELISA kit. Phospholipase C (PLC)/ Protein kinase C (PKC) signaling pathway was analyzed by a Western blot technique. The effect of the SCED was also studied in vivo on bleeding time in mice. ResultsSCED improved the neurological defect score, increased cerebral blood flow, reduced infarct size and alleviated brain edema in rats exposed to pMCAO. After administration of SCED, thrombosis formation in arteriovenous shunt was inhibited and recovery time in pulmonary embolism was shortened. The inhibitory effect of SCED on platelet activation was further confirmed by TXB2 ELISA kit and Western blot analysis of PLC/PKC signaling pathway. ConclusionsSCED attenuates cerebral ischemic injury. The possible mechanism is that SCED inhibits thrombosis formation, platelet aggregation and activation of PLC/PKC pathway. On this basis, this new extract could be a promising agent to inhibit thrombosis formation and protect against cerebral ischemia injury.

Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.