RESUMEN
Background/aim: Our recent study revealed that the expression of lipoxygenase (LOX) and cyclooxygenase (COX) enzymes in the hypothalamus is activated by nesfatin-1, leading to the liberation of leukotrienes and prostaglandins (PG), respectively. Moreover, our prior report explained that intracerebroventricular (ICV) nesfatin-1 treatment triggers cardiovascular responses mediated by central LOX and COX enzymes. Building upon our prior reports, the present investigation sought to clarify the role of cardiovascularly active central COX products, such as thromboxane (TX) A2, PGF2α, PGE, and PGD, in orchestrating nesfatin-1-evoked reactions in mean arterial pressure (MAP) and heart rate (HR). Materials and methods: The Sprague Dawley rats, which had guide cannula in the lateral ventricle for intracerebroventricular (ICV) injections and catheter in arteria femoralis for monitoring MAP and HR, were underwent central pretreatment with furegrelate (the TXA2 synthase inhibitor), PGF2α-dimethylamine (PGF2α-DA, the PGF2α receptor antagonist), or AH6809 (the PGE and PGD receptor antagonist), 5 min prior to ICV nesfatin-1 administration. The cardiovascular parameters were observed and recorded for 60 min posttreatment. Results: Nesfatin-1 induced cardiovascular responses in rats leading to pressor effect in MAP, and tachycardia following bradycardia in HR. Interestingly, ICV furegrelate, PGF2α-DA, or AH6809 pretreatment partially mitigated the cardiovascular effects revealed by nesfatin-1. Conclusion: The findings illuminate the role of nesfatin-1 in modulating MAP and HR through the central activation of specifically TXA2, PGF2α, PGE, and PGD from COX metabolites. Additionally, the study may also suggest the potential involvement of other central COX or LOX metabolites beyond these COX metabolites in mediating the cardiovascular effects produced by nesfatin-1.
Asunto(s)
Nucleobindinas , Ratas Sprague-Dawley , Tromboxano A2 , Animales , Nucleobindinas/farmacología , Ratas , Masculino , Tromboxano A2/metabolismo , Dinoprost/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Dinoprostona/farmacología , Dinoprostona/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/farmacología , Presión Sanguínea/efectos de los fármacosRESUMEN
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.
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Aorta , Lisofosfolípidos , Ratones Endogámicos C57BL , Tromboxano A2 , Vasoconstricción , Animales , Tromboxano A2/metabolismo , Vasoconstricción/efectos de los fármacos , Ratones , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Masculino , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Calcio/metabolismoRESUMEN
BACKGROUND: Experimental preeclampsia (ePE) has been shown to have worsened outcome from stroke. We investigated the effect of low-dose aspirin, known to prevent preeclampsia, on stroke hemodynamics and outcome, and the association between the vasoconstrictor and vasodilator cyclooxygenase products thromboxane A2 and prostacyclin. METHODS AND RESULTS: Middle cerebral artery occlusion was performed for 3 hours with 1 hour of reperfusion in normal pregnant rats on day 20 of gestation and compared with ePE treated with vehicle or low-dose aspirin (1.5 mg/kg per day). Multisite laser Doppler was used to measure changes in cerebral blood flow to the core middle cerebral artery and collateral vascular territories. After 30 minutes occlusion, phenylephrine was infused to increase blood pressure and assess cerebral blood flow autoregulation. Infarct and edema were measured using 2,3,5-triphenyltetrazolium chloride staining. Plasma levels of thromboxane A2, prostacyclin, and inflammatory markers in plasma and cyclooxygenase levels in cerebral arteries were measured. ePE had increased infarction compared with normal pregnant rats (P<0.05) that was reduced by aspirin (P<0.001). ePE also had intact cerebral blood flow autoregulation and reduced collateral perfusion during induced hypertension that was also prevented by aspirin. Aspirin increased prostacyclin in ePE (P<0.05) without reducing thromboxane B2, metabolite of thromboxane A2, or 8-isoprostane-prostaglandin-2α, a marker of lipid peroxidation. There were no differences in cyclooxygenase levels in cerebral arteries between groups. CONCLUSIONS: Low-dose aspirin in ePE reduced infarction that was associated with increased vasodilator prostacyclin and improved collateral perfusion during induced hypertension. The beneficial effect of aspirin on the brain and cerebral circulation is likely multifactorial and worth further study.
Asunto(s)
Aspirina , Circulación Cerebrovascular , Circulación Colateral , Modelos Animales de Enfermedad , Homeostasis , Preeclampsia , Ratas Sprague-Dawley , Animales , Femenino , Embarazo , Aspirina/administración & dosificación , Aspirina/farmacología , Circulación Cerebrovascular/efectos de los fármacos , Preeclampsia/fisiopatología , Preeclampsia/metabolismo , Preeclampsia/tratamiento farmacológico , Homeostasis/efectos de los fármacos , Circulación Colateral/efectos de los fármacos , Tromboxano A2/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Ratas , Epoprostenol/metabolismo , Flujometría por Láser-DopplerRESUMEN
Acquired disorders of platelet function are an underdiagnosed cause of bleeding tendency. A 14-year-old girl developed moderate mucocutaneous bleeding two weeks after a Mycoplasma pneumoniae infection successfully treated with clarithromycin. The patient was referred to us 7 months later for laboratory investigation of the persisting bleeding diathesis. The patient's personal and family histories were negative for bleeding disorders. Complete blood count, von Willebrand Factor levels and coagulation tests were normal; platelet aggregation, ATP secretion, δ-granules content and serum thromboxane B2 levels were defective. At follow-up visits, laboratory parameters and the bleeding diathesis progressively normalized within 2 years. The patient's condition is compatible with a diagnosis of acquired Storage Pool Deficiency (SPD), associated with defective thromboxane A2 production. To our knowledge, this is the first case of acquired, transient SPD with spontaneous remission. The pathogenic role of Mycoplasma pneumoniae infection or clarithromycin is possible, albeit uncertain.
Asunto(s)
Deficiencia de Almacenamiento del Pool Plaquetario , Tromboxano A2 , Humanos , Femenino , Adolescente , Deficiencia de Almacenamiento del Pool Plaquetario/complicaciones , Tromboxano A2/metabolismo , Plaquetas/metabolismo , Trastornos HemorrágicosRESUMEN
BACKGROUND: Prenatal hypoxia, a common pregnancy complication, leads to impaired cardiovascular outcomes in the adult offspring. It results in impaired vasodilation in coronary and mesenteric arteries of the adult offspring, due to reduced nitric oxide (NO). Thromboxane A2 (TxA2) is a potent vasoconstrictor increased in cardiovascular diseases, but its role in the impact of prenatal hypoxia is unknown. To prevent the risk of cardiovascular disease by prenatal hypoxia, we have tested a maternal treatment using a nanoparticle-encapsulated mitochondrial antioxidant (nMitoQ). We hypothesized that prenatal hypoxia enhances vascular TxA2 responses in the adult offspring, due to decreased NO modulation, and that this might be prevented by maternal nMitoQ treatment. METHODS: Pregnant Sprague-Dawley rats received a single intravenous injection (100 µL) of vehicle (saline) or nMitoQ (125 µmol/L) on gestational day (GD)15 and were exposed to normoxia (21% O2) or hypoxia (11% O2) from GD15 to GD21 (term = 22 days). Coronary and mesenteric arteries were isolated from the 4-month-old female and male offspring, and vasoconstriction responses to U46619 (TxA2 analog) were evaluated using wire myography. In mesenteric arteries, L-NAME (pan-NO synthase (NOS) inhibitor) was used to assess NO modulation. Mesenteric artery endothelial (e)NOS, and TxA2 receptor expression, superoxide, and 3-nitrotyrosine levels were assessed by immunofluorescence. RESULTS: Prenatal hypoxia resulted in increased U46619 responsiveness in coronary and mesenteric arteries of the female offspring, and to a lesser extent in the male offspring, which was prevented by nMitoQ. In females, there was a reduced impact of L-NAME in mesenteric arteries of the prenatal hypoxia saline-treated females, and reduced 3-nitrotyrosine levels. In males, L-NAME increased U46619 responses in mesenteric artery to a similar extent, but TxA2 receptor expression was increased by prenatal hypoxia. There were no changes in eNOS or superoxide levels. CONCLUSIONS: Prenatal hypoxia increased TxA2 vasoconstrictor capacity in the adult offspring in a sex-specific manner, via reduced NO modulation in females and increased TP expression in males. Maternal placental antioxidant treatment prevented the impact of prenatal hypoxia. These findings increase our understanding of how complicated pregnancies can lead to a sex difference in the programming of cardiovascular disease in the adult offspring.
Prenatal hypoxia, when the fetus does not receive enough oxygen, is a common problem during pregnancy that impacts the developing fetus. It is associated with an increased risk of cardiovascular disease in the offspring in adulthood. While the mechanisms are not fully understood, the blood vessel function in the offspring may be impacted by prenatal hypoxia. We hypothesize that prenatal hypoxia increases the constriction of the blood vessels in the offspring. The placenta, an essential organ for fetal development, supplies oxygen and nutrients to the fetus. In prenatal hypoxia pregnancies, the placenta does not work properly. We have been studying a placental treatment (called nMitoQ) to improve placenta function and thereby the blood vessel function of the offspring. We used a rat model of prenatal hypoxia, where pregnant rats (dams) were placed in a low oxygen environment (hypoxia) during the last trimester of pregnancy. Control rats were kept in normal oxygen conditions. The dams were treated with nMitoQ, or with saline (control). Next, we studied the blood vessels of the offspring in adulthood. We found that prenatal hypoxia increases the constriction of the blood vessels, which was prevented by treating the dams with nMitoQ. Interestingly, this impact was more severe in females compared to males, and the mechanisms were different between the sexes. This study helps in the understanding of how complicated pregnancies can impair cardiovascular health in the offspring, and in a potential development of targeted and sex-specific therapies for those offspring at high risk for future cardiovascular disease.
Asunto(s)
Efectos Tardíos de la Exposición Prenatal , Ratas Sprague-Dawley , Caracteres Sexuales , Tromboxano A2 , Vasoconstricción , Animales , Femenino , Embarazo , Vasoconstricción/efectos de los fármacos , Masculino , Tromboxano A2/metabolismo , Antioxidantes/farmacología , Óxido Nítrico/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Ratas , Hipoxia/metabolismo , Hipoxia Fetal/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologíaRESUMEN
Prostanoids are endogenous lipid bioactive mediators that play essential roles in physiological processes such as glucocorticoid secretion. Here, it is found that the thromboxane (Tx)A2 receptor (TP) is highly expressed in the adrenal cortex of mice. Both global and adrenocortical-specific deletion of the TP receptor lead to increased adiposity in mice by elevating corticosterone synthesis. Mechanistically, the TP receptor deletion increases the phosphorylation of steroidogenic acute regulatory protein (StAR) and corticosterone synthesis in adrenal cortical cells by suppressing p-p38-mediated phosphorylation of 14-3-3γ adapter protein at S71. The activation of the p38 in the adrenal cortical cells by forced expression of the MKK6EE gene attenuates hypercortisolism in TP-deficient mice. These observations suggest that the TxA2/TP signaling regulates adrenal corticosterone homeostasis independent of the hypothalamic-pituitary-adrenal axis and the TP receptor may serve as a promising therapeutic target for hypercortisolism.
Asunto(s)
Corticosterona , Fosfoproteínas , Receptores de Tromboxano A2 y Prostaglandina H2 , Tromboxano A2 , Animales , Masculino , Ratones , Corteza Suprarrenal/metabolismo , Corticosterona/metabolismo , Ratones Endogámicos C57BL , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Transducción de Señal , Tromboxano A2/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
In lung, thromboxane A2 (TXA2) activates the TP receptor to induce proinflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased approximately 2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naive CD4+ T cell differentiation to Th9 cells and IL-9 production were inhibited dose-dependently by TXA2 in vitro. TP receptor-deficient mice had an approximately 2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared with wild-type mice. Naive CD4+ T cells from TP-deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared with CD4+ T cells from wild-type mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, proinflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.
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Asma , Diferenciación Celular , Linfocitos T Reguladores , Células Th2 , Tromboxano A2 , Animales , Ratones , Células Th2/inmunología , Células Th2/patología , Tromboxano A2/metabolismo , Tromboxano A2/inmunología , Linfocitos T Reguladores/inmunología , Asma/inmunología , Asma/patología , Asma/tratamiento farmacológico , Asma/genética , Ratones Noqueados , Interleucina-9/inmunología , Interleucina-9/genética , Interleucina-9/metabolismo , Neumonía/inmunología , Neumonía/patología , Ratones Endogámicos C57BL , Ratones Endogámicos BALB C , Pulmón/inmunología , Pulmón/patología , Ovalbúmina/inmunología , Femenino , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.
Asunto(s)
Eugenol , Embolia Pulmonar , Humanos , Ratones , Animales , Eugenol/farmacología , Eugenol/uso terapéutico , Eugenol/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Modelos Animales de Enfermedad , Activación Plaquetaria , Agregación Plaquetaria , Plaquetas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Tromboxano A2/metabolismo , Colágeno/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/metabolismo , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
BACKGROUND: Vascular injury results in uncontrollable hemorrhage in hemorrhagic diseases and excessive antithrombotic therapy. Safe and efficient hemostatic agents which can be orally administered are urgently needed. Platelets play indispensable roles in hemostasis, but there is no drug exerting hemostatic effects through enhancing platelet function. METHODS: The regulatory effects of icaritin, a natural compound isolated from Herba Epimedii, on the dense granule release, thromboxane A2 (TxA2) synthesis, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by multiple agonists were investigated. The effects of icaritin on tail vein bleeding times of warfarin-treated mice were also evaluated. Furthermore, we investigated the underlying mechanisms by which icaritin exerted its pharmacological effects. RESULTS: Icaritin alone did not activate platelets, but significantly potentiated the dense granule release, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by thrombin and U46619. Icaritin also shortened tail vein bleeding times of mice treated with warfarin. In addition, phosphorylated proteome analysis, immunoblotting analysis, and pharmacological research revealed that icaritin sensitized the activation of phospholipase Cγ2 (PLCγ2)-protein kinase C (PKC) signaling pathways, which play important roles in platelet activation. CONCLUSION: Icaritin can sensitize platelet activation induced by thrombin and TxA2 through enhancing the activation of PLCγ2-PKC signaling pathways and promote hemostasis, and has potential to be developed into a novel orally deliverable therapeutic agent for hemorrhages.
Asunto(s)
Plaquetas , Flavonoides , Hemostasis , Fosfolipasa C gamma , Activación Plaquetaria , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Proteína Quinasa C , Transducción de Señal , Trombina , Tromboxano A2 , Animales , Fosfolipasa C gamma/metabolismo , Transducción de Señal/efectos de los fármacos , Tromboxano A2/metabolismo , Activación Plaquetaria/efectos de los fármacos , Hemostasis/efectos de los fármacos , Trombina/metabolismo , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Proteína Quinasa C/metabolismo , Flavonoides/farmacología , Ratones , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Hemorragia/inducido químicamente , Hemorragia/tratamiento farmacológico , Tiempo de SangríaRESUMEN
Asthma is the chronic pulmonary inflammatory response that could lead to respiratory failure when allergic reactions exacerbate. It is featured by type 2 immunity with eosinophilic inflammation, mucus, and IgE production, and Th2 cytokine secretion upon repeated challenge of allergens. The symptom severity of asthma displays an apparent circadian rhythm with aggravated airway resistance in the early morning in patients. Bmal1 is the core regulator of the circadian clock, while the regulatory role of Bmal1 in asthma remains unclear. Here, we investigate whether the myeloid Bmal1 is involved in the pathogenesis of house dust mite (HDM)-induced lung allergy. We found that knockdown of Bmal1 in macrophages suppressed the time-of-day variance of the eosinophil infiltration in the alveolar spaces in chronic asthmatic mice. This was accompanied by decreased bronchial mucus production, collagen deposition, and HDM-specific IgE production. However, the suppression effects of myeloid Bmal1 deletion did not alter the allergic responses in short-term exposure to HDM. The transcriptome profile of alveolar macrophages (AMs) showed that Bmal1-deficient AMs have enhanced phagocytosis and reduced production of allergy-mediating prostanoids thromboxane A2 and prostaglandin F2α synthesis. The attenuated thromboxane A2 and prostaglandin F2α may lead to less induction of the eosinophil chemokine Ccl11 expression in bronchial epithelial cells. In summary, our study demonstrates that Bmal1 ablation in macrophages attenuates eosinophilic inflammation in HDM-induced chronic lung allergy, which involves enhanced phagocytosis and reduced prostanoid secretion.
Asunto(s)
Asma , Eosinofilia , Hipersensibilidad , Humanos , Ratones , Animales , Pyroglyphidae , Dinoprost/metabolismo , Tromboxano A2/metabolismo , Pulmón , Alérgenos , Eosinofilia/metabolismo , Eosinofilia/patología , Inmunoglobulina E/metabolismo , Inflamación/patología , Modelos Animales de EnfermedadRESUMEN
BACKGROUND AND PURPOSE: Defective insulin signalling and dysfunction of the endoplasmic reticulum (ER), driven by excessive lipid accumulation in the liver, is a characteristic feature in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Thromboxane A2 (TXA2 ), an arachidonic acid metabolite, is significantly elevated in obesity and plays a crucial role in hepatic gluconeogenesis and adipose tissue macrophage polarization. However, the role of liver TXA2 /TP receptors in insulin resistance and lipid metabolism is largely unknown. EXPERIMENTAL APPROACH: TP receptor knockout (TP-/- ) mice were generated and fed a high-fat diet for 16 weeks. Insulin sensitivity, ER stress responses and hepatic lipid accumulation were assessed. Furthermore, we used primary hepatocytes to dissect the mechanisms by which the TXA2 /TP receptor axis regulates insulin signalling and hepatocyte lipogenesis. KEY RESULTS: TXA2 was increased in diet-induced obese mice, and depletion of TP receptors in adult mice improved systemic insulin resistance and hepatic steatosis. Mechanistically, we found that the TXA2 /TP receptor axis disrupts insulin signalling by activating the Ca2+ /calcium calmodulin-dependent kinase II γ (CaMKIIγ)-protein kinase RNA-like endoplasmic reticulum kinase (PERK)-C/EBP homologous protein (Chop)-tribbles-like protein 3 (TRB3) axis in hepatocytes. In addition, our results revealed that the TXA2 /TP receptor axis directly promoted lipogenesis in primary hepatocytes and contributed to Kupffer cell inflammation. CONCLUSIONS AND IMPLICATIONS: The TXA2 /TP receptor axis facilitates insulin resistance through Ca2+ /CaMKIIγ to activate PERK-Chop-TRB3 signalling. Inhibition of hepatocyte TP receptors improved hepatic steatosis and inflammation. The TP receptor is a new therapeutic target for NAFLD and metabolic syndrome.
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Resistencia a la Insulina , Insulinas , Enfermedad del Hígado Graso no Alcohólico , Animales , Ratones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/uso terapéutico , Tromboxano A2/metabolismo , Tromboxano A2/uso terapéutico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Estrés del Retículo Endoplásmico , Insulinas/metabolismo , Dieta Alta en Grasa , Ratones Endogámicos C57BLRESUMEN
INTRODUCTION: Cardioplegia and cardiopulmonary bypass (CP/CPB) alters coronary arteriolar response to thromboxane A2 (TXA2) in patients undergoing cardiac surgery. Comorbidities, including hypertension (HTN), can further alter coronary vasomotor tone. This study investigates the effects of HTN on coronary arteriolar response to TXA2 pre and post-CP/CPB and cardiac surgery. MATERIALS AND METHODS: Coronary arterioles pre and post-CP/CPB were dissected from atrial tissue samples in patients with no HTN (NH, n = 9), well-controlled HTN (WC, n = 12), or uncontrolled HTN (UC, n = 12). In-vitro coronary microvascular reactivity was examined in the presence of TXA2 analog U46619 (10-9-10-4M). Protein expression of TXA2 receptor in the harvested right atrial tissue samples were measured by immunoblotting. RESULTS: TXA2 analog U46619 induced dose-dependent contractile responses of coronary arterioles in all groups. Pre-CPB contractile responses to U46619 were significantly increased in microvessels in the UC group compared to the NH group (P < 0.05). The pre-CP/CPB contractile responses of coronary arterioles were significantly diminished post-CP/CPB among the three groups (P < 0.05), but there remained an increased contractile response in the microvessels of the UC group compared to the WC and NH groups (P < 0.05). There were no significant differences in U46619-induced vasomotor tone between patients in the NH and WC groups (P > 0.05). There were no differences in expression of TXA2R among groups. CONCLUSIONS: Poorly controlled HTN is associated with increased contractile response of coronary arterioles to TXA2. This alteration may contribute to worsened recovery of coronary microvascular function in patients with poorly controlled HTN after CP/CPB and cardiac surgery.
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Fibrilación Atrial , Procedimientos Quirúrgicos Cardíacos , Hipertensión , Humanos , Tromboxano A2/metabolismo , Tromboxano A2/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Vasos Coronarios , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Puente Cardiopulmonar , Hipertensión/complicacionesRESUMEN
In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.
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Megacariocitos , Tromboxanos , Humanos , Megacariocitos/metabolismo , Preparaciones de Acción Retardada , Plaquetas/metabolismo , Proteínas de Unión al GTP/metabolismo , Tromboxano A2/metabolismoRESUMEN
Over the last two decades, awareness of the (patho)physiological roles of thromboxane A2 signaling has been greatly extended. From humble beginnings as a short-lived stimulus that activates platelets and causes vasoconstriction to a dichotomous receptor system involving multiple endogenous ligands capable of modifying tissue homeostasis and disease generation in almost every tissue of the body. Thromboxane A2 receptor (TP) signal transduction is associated with the pathogenesis of cancer, atherosclerosis, heart disease, asthma, and host response to parasitic infection amongst others. The two receptors mediating these cellular responses (TPα and TPß) are derived from a single gene (TBXA2R) through alternative splicing. Recently, knowledge about the mechanism(s) of signal propagation by the two receptors has undergone a revolution in understanding. Not only have the structural relationships associated with G-protein coupling been established but the modulation of that signaling by post-translational modification to the receptor has come sharply into focus. Moreover, the signaling of the receptor unrelated to G-protein coupling has become a burgeoning field of endeavor with over 70 interacting proteins currently identified. These data are reshaping the concept of TP signaling from a mere guanine nucleotide exchange factors for Gα activation to a nexus for the convergence of diverse and poorly characterized signaling pathways. This review summarizes the advances in understanding in TP signaling, and the potential for new growth in a field that after almost 50 years is finally coming of age.
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Transducción de Señal , Tromboxanos , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Tromboxano A2/metabolismoRESUMEN
Obesity is a complex disease highly related to diet and lifestyle and is associated with low amount of thermogenic adipocytes. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to fight overweight and associated comorbidities. Recent studies suggest a role for several fatty acids and their metabolites, called lipokines, in the control of thermogenesis. The purpose of this work was to analyze the role of several lipokines in the control of brown/brite adipocyte formation. We used a validated human adipocyte model, human multipotent adipose-derived stem cell model (hMADS). In the absence of rosiglitazone, hMADS cells differentiate into white adipocytes, but convert into brite adipocytes upon rosiglitazone or prostacyclin 2 (PGI2) treatment. Gene expression was quantified using RT-qPCR and protein levels were assessed by Western blotting. We show here that lipokines such as 12,13-diHOME, 12-HEPE, 15dPGJ2 and 15dPGJ3 were not able to induce browning of white hMADS adipocytes. However, both fatty acid esters of hydroxy fatty acids (FAHFAs), 9-PAHPA and 9-PAHSA potentiated brown key marker UCP1 mRNA levels. Interestingly, CTA2, the stable analog of thromboxane A2 (TXA2), but not its inactive metabolite TXB2, inhibited the rosiglitazone and PGI2-induced browning of hMADS adipocytes. These results pinpoint TXA2 as a lipokine inhibiting brown adipocyte formation that is antagonized by PGI2. Our data open new horizons in the development of potential therapies based on the control of thromboxane A2/prostacyclin balance to combat obesity and associated metabolic disorders.
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Ácidos Grasos , Tromboxano A2 , Humanos , Tromboxano A2/metabolismo , Rosiglitazona/farmacología , Ácidos Grasos/metabolismo , Adipocitos Marrones/metabolismo , Obesidad/metabolismo , Prostaglandinas I/metabolismoRESUMEN
Excessive hepatic glucose production (HGP) is a major cause of fasting hyperglycemia in diabetes, and antihyperglycemic therapy takes center stage. Nonsteroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), reduce hyperglycemia caused by unrestrained gluconeogenesis in diabetes, but its mechanism is incompletely understood. Here, we reported that aspirin lowers fasting blood glucose and hepatic gluconeogenesis, corresponds with lower thromboxane A2 (TXA2) levels, and the hypoglycemic effect of aspirin could be rescued by TP agonist treatment. On fasting and diabetes stress, the cyclooxygenase (COX)/TXA2/thromboxane A2 receptor (TP) axis was increased in the livers. TP deficiency suppressed starvation-induced hepatic glucose output, thus inhibiting the progression of diabetes, whereas TP activation promoted gluconeogenesis. Aspirin restrains glucagon signaling and gluconeogenic gene expression (phosphoenolpyruvate carboxykinase [PCK1] and glucose-6-phosphatase [G6Pase]) through the TXA2/TP axis. TP mediates hepatic gluconeogenesis by activating PLC/IP3/IP3R signaling, which subsequently enhances CREB phosphorylation via facilitating CRTC2 nuclear translocation. Thus, our findings demonstrate that TXA2/TP plays a crucial role in aspirin's inhibition of hepatic glucose metabolism, and TP may represent a therapeutic target for diabetes.
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Diabetes Mellitus , Hiperglucemia , Humanos , Glucagón/metabolismo , Tromboxano A2/metabolismo , Aspirina/farmacología , Aspirina/metabolismo , Hígado/metabolismo , Glucosa/metabolismo , Gluconeogénesis , Diabetes Mellitus/metabolismo , Hipoglucemiantes , Hiperglucemia/metabolismoRESUMEN
Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.
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Plaquetas , Calcio , Proteína ORAI1 , Animales , Femenino , Masculino , Ratones , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Plaquetas/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Colágeno/metabolismo , Proteína ORAI1/metabolismo , Péptidos/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Tapsigargina/farmacología , Trombina/farmacología , Tromboxano A2/metabolismoRESUMEN
Low-dose aspirin is currently recommended for patients with polycythemia vera (PV), a myeloproliferative neoplasm with increased risk of arterial and venous thromboses. Based on aspirin pharmacodynamics in essential thrombocythemia, a twice-daily regimen is recommended for patients with PV deemed at particularly high thrombotic risk. We investigated the effects of low-dose aspirin on platelet cyclooxygenase activity and in vivo platelet activation in 49 patients with PV, as assessed by serum thromboxane (TX) B2 and urinary TXA2 /TXB2 metabolite (TXM) measurements, respectively. A previously described pharmacokinetic-pharmacodynamic in silico model was used to simulate the degree of platelet TXA2 inhibition by once-daily (q.d.) and twice-daily (b.i.d.) aspirin, and to predict the effect of missing an aspirin dose during q.d. and b.i.d. regimens. Serum TXB2 averaged 8.2 (1.6-54.7) ng/ml and significantly correlated with the platelet count (γ = 0.39) and urinary TXM (γ = 0.52) in multivariable analysis. One-third of aspirin-treated patients with PV displayed less-than-maximal platelet TXB2 inhibition, and were characterized by significantly higher platelet counts and platelet-count corrected serum TXB2 than those with adequate inhibition. Eight patients with PV were sampled again after 12 ± 4 months, and had reproducible serum TXB2 and urinary TXM values. The in silico model predicted complete inhibition of platelet-derived TXB2 by b.i.d. aspirin, a prediction verified in a patient with PV with the highest TXB2 value while on aspirin q.d. and treated short-term with a b.i.d. regimen. In conclusion, one in three patients with PV on low-dose aspirin display less-than-maximal inhibition of platelet TXA2 production. Serum TXB2 measurement can be a valuable option to guide precision dosing of antiplatelet therapy in patients with PV.
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Policitemia Vera , Humanos , Policitemia Vera/tratamiento farmacológico , Policitemia Vera/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacología , Tromboxanos/uso terapéutico , Aspirina/farmacología , Plaquetas/metabolismo , Tromboxano B2 , Tromboxano A2/metabolismo , Tromboxano A2/farmacología , Simulación por Computador , Inhibidores de Agregación PlaquetariaRESUMEN
In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones (Klf15 and Spp1). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.
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Receptores ErbB/metabolismo , Músculo Liso Vascular , Miocitos del Músculo Liso , Receptor de Angiotensina Tipo 1/metabolismo , Tromboxano A2/metabolismo , Angiotensina II/metabolismo , Animales , Receptores ErbB/genética , Regulación de la Expresión Génica , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.