RESUMO
BACKGROUND: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. METHODS: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. RESULTS: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. CONCLUSIONS: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.
Assuntos
COVID-19 , Trombose , Humanos , Anticorpos Monoclonais/farmacologia , Plaquetas/metabolismo , COVID-19/metabolismo , Estudos Transversais , SARS-CoV-2 , Trombose/etiologia , Trombose/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacologia , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND AND AIMS: Thromboxane (TX) A2, released by activated platelets, plays an important role in atherothrombosis. Urinary 11-dehydro-TXB2 (U-TXM), a stable metabolite reflecting the whole-body TXA2 biosynthesis, is reduced by â¼70% by daily low-dose aspirin. The U-TXM represents a non-invasive biomarker of in vivo platelet activation and is enhanced in patients with diabetes. This study assessed whether U-TXM is associated with the risk of future serious vascular events or revascularizations (SVE-R), major bleeding, or cancer in patients with diabetes. METHODS: The U-TXM was measured pre-randomization to aspirin or placebo in 5948 people with type 1 or 2 diabetes and no cardiovascular disease, in the ASCEND trial. Associations between log U-TXM and SVE-R (n = 618), major bleed (n = 206), and cancer (n = 700) during 6.6 years of follow-up were investigated by Cox regression; comparisons of these associations with the effects of randomization to aspirin were made. RESULTS: Higher U-TXM was associated with older age, female sex, current smoking, type 2 diabetes, higher body size, urinary albumin/creatinine ratio of ≥3 mg/mmol, and higher estimated glomerular filtration rate. After adjustment for these, U-TXM was marginally statistically significantly associated with SVE-R and major bleed but not cancer [hazard ratios per 1 SD higher log U-TXM (95% confidence interval): 1.09 (1.00-1.18), 1.16 (1.01-1.34), and 1.06 (0.98-1.14)]. The hazard ratio was similar to that implied by the clinical effects of randomization to aspirin for SVE-R but not for major bleed. CONCLUSIONS: The U-TXM was log-linearly independently associated with SVE-R in diabetes. This is consistent with the involvement of platelet TXA2 in diabetic atherothrombosis.
Assuntos
Diabetes Mellitus Tipo 2 , Neoplasias , Trombose , Humanos , Feminino , Tromboxanos/metabolismo , Tromboxanos/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Aspirina/uso terapêutico , Tromboxano B2/uso terapêutico , Tromboxano B2/urina , Tromboxano A2/uso terapêutico , Tromboxano A2/urina , Trombose/tratamento farmacológico , Neoplasias/tratamento farmacológicoRESUMO
BACKGROUND: Systemic thromboxane A2 generation, assessed by quantifying the concentration of stable thromboxane B2 metabolites (TXB2-M) in the urine adjusted for urinary creatinine, is strongly associated with mortality risk. We sought to define optimal TXB2-M cutpoints for aspirin users and nonusers and determine if adjusting TXB2-M for estimated glomerular filtration rate (eGFR) in addition to urinary creatinine improved mortality risk assessment. METHODS: Urinary TXB2-M were measured by competitive ELISA in 1363 aspirin users and 1681 nonusers participating in the Framingham Heart Study. Cutpoints were determined for TXB2-M and TXB2-M/eGFR using log-rank statistics and used to assess mortality risk by Cox proportional hazard modeling and restricted mean survival time. Multivariable models were compared using the Akaike Information Criterion (AIC). A cohort of 105 aspirin users with heart failure was used for external validation. RESULTS: Optimized cutpoints of TXB2-M were 1291 and 5609â pg/mg creatinine and of TXB2-M/eGFR were 16.6 and 62.1 filtered prostanoid units (defined as pg·min/creatinine·mL·1.73â m2), for aspirin users and nonusers, respectively. TXB2-M/eGFR cutpoints provided more robust all-cause mortality risk discrimination than TXB2-M cutpoints, with a larger unadjusted hazard ratio (2.88 vs 2.16, AIC P < 0.0001) and greater differences in restricted mean survival time between exposure groups (1.46 vs 1.10 years), findings that were confirmed in the external validation cohort of aspirin users. TXB2-M/eGFR cutpoints also provided better cardiovascular/stroke mortality risk discrimination than TXB2-M cutpoints (unadjusted hazard ratio 3.31 vs 2.13, AIC P < 0.0001). CONCLUSION: Adjustment for eGFR strengthens the association of urinary TXB2-M with long-term mortality risk irrespective of aspirin use.
Assuntos
Aspirina , Tromboxanos , Humanos , Prognóstico , Creatinina/urina , Aspirina/uso terapêutico , Tromboxano B2/metabolismo , Rim/metabolismoRESUMO
BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbß3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.
Assuntos
Araquidonato 12-Lipoxigenase , Plaquetas , Humanos , Camundongos , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Plaquetas/metabolismo , Camundongos Transgênicos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Tromboxanos/metabolismoRESUMO
Despite decades of investigations, the optimal assessment of the "therapeutic response" to early after loading dose of acetylsalicylic acid (ASA) remains unclear. Limited information is available on the relation between pharmacodynamic (PD) and pharmacokinetic (PK) measurements assessed immediately after ASA administration. Serial PD and PK analyses were performed immediately after a single 162 or 650 mg dose of chewed and swallowed ASA in ten healthy adults. ASA response was defined as > 95% inhibition of serum thromboxane (Tx)B2, < 550 aspirin reaction units (ARU) by VerifyNow Aspirin (VN) test, and ≤ 20% arachidonic acid (AA)-induced platelet aggregation (PA). Correlation analyses between PK and PD measurements and receiver operating characteristic (ROC) curve analyses were performed. ASA response measured by VN test and AA-induced PA was achieved within 30 min of ASA administration. A correlation was observed between ARU and AA-induced maximum PA (r = 0.69, p < 0.001), serum TxB2 (r = 0.74 and p < 0.001), and serum TxB2 inhibition (r = 0.79, p < 0.001). In ROC curve analyses, ≤ 558 ARU and ≤ 7% AA-induced PA were associated with > 95% inhibition of TxB2. 686 ng/ml plasma ASA cut-off point was associated with > 95% inhibition of serum TxB2, ≤ 7% 1 mM AA-induced PA, and ≤ 585 ARU. A modest ~ 50% inhibition of TxB2 inhibition was associated with marked inhibition of 1 mM AA-induced platelet aggregation by LTA. Our analyses demonstrated important relationships between pharmacodynamic, and pharmacokinetic parameters measured immediately following oral ASA and cutoff values for ARU and AA-induced PA that is associated with > 95% inhibition of serum TxB2.
Assuntos
Aspirina , Inibidores da Agregação Plaquetária , Adulto , Humanos , Aspirina/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Tromboxano B2 , Agregação Plaquetária , Tromboxanos , Ácido Araquidônico/farmacologia , PlaquetasRESUMO
BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is the triad of asthma, nasal polyposis, and respiratory reactions to COX-1 inhibitors. Overproduction of cysteinyl leukotrienes and underproduction of prostaglandin E2 (PGE2) are hallmarks of AERD. A mouse model predicted a key role for the thromboxane-prostanoid (TP) receptor in AERD. OBJECTIVE: Our aim was to determine whether ifetroban, a TP receptor antagonist, attenuates aspirin-induced respiratory symptoms in patients with AERD. METHODS: A total of 35 patients with AERD completed a 4-week double-blinded, placebo-controlled trial of ifetroban and underwent an oral aspirin challenge. The primary outcome was change in the provocative dose of aspirin that caused a 2-point increase in Total Nasal Symptom Score. Changes in lung function, eicosanoid levels, and platelet and mast cell activation were assessed. Cultured human nasal fibroblasts were stimulated with or without the TP agonist U46619 and assayed for prostanoid production. RESULTS: Ifetroban was well tolerated in AERD and did not change the mean 2-point increase in Total Nasal Symptom Score (P = .763). Participants taking ifetroban had greater aspirin-induced nasal symptoms and a greater decline in FEV1 value than did participants receiving placebo (-18.8% ± 3.6% with ifetroban vs -8.4% ± 2.1% with placebo [P = .017]). Four weeks of ifetroban significantly increased urinary leukotriene E4 levels and decreased nasal PGE2 levels compared with placebo. Peak aspirin-induced urinary thromboxane levels correlated with peak urinary leukotriene E4 and prostaglandin D2 metabolite levels in participants taking ifetroban. U46119 significantly potentiated the production of PGE2 by cultured nasal fibroblasts from subjects with AERD but not by cultured nasal fibroblasts from controls without polypoid sinusitis. CONCLUSION: Contrary to our hypothesis, TP receptor blockade worsened aspirin-induced reactions in AERD, possibly by exacerbating dysregulation of the eicosanoid system. TP signaling on stromal cells may be critical to maintaining PGE2 production when COX-2 function is low.
Assuntos
Asma Induzida por Aspirina , Sinusite , Animais , Camundongos , Humanos , Prostaglandinas , Tromboxanos/uso terapêutico , Leucotrieno E4 , Receptores de Tromboxanos/uso terapêutico , Asma Induzida por Aspirina/tratamento farmacológico , Asma Induzida por Aspirina/diagnóstico , Aspirina/efeitos adversos , Eicosanoides , Dinoprostona , Homeostase , Sinusite/induzido quimicamenteRESUMO
BACKGROUND: Pre-clinical models demonstrate that platelet activation is involved in the spread of malignancy. Ongoing clinical trials are assessing whether aspirin, which inhibits platelet activation, can prevent or delay metastases. METHODS: Urinary 11-dehydro-thromboxane B2 (U-TXM), a biomarker of in vivo platelet activation, was measured after radical cancer therapy and correlated with patient demographics, tumour type, recent treatment, and aspirin use (100 mg, 300 mg or placebo daily) using multivariable linear regression models with log-transformed values. RESULTS: In total, 716 patients (breast 260, colorectal 192, gastro-oesophageal 53, prostate 211) median age 61 years, 50% male were studied. Baseline median U-TXM were breast 782; colorectal 1060; gastro-oesophageal 1675 and prostate 826 pg/mg creatinine; higher than healthy individuals (~500 pg/mg creatinine). Higher levels were associated with raised body mass index, inflammatory markers, and in the colorectal and gastro-oesophageal participants compared to breast participants (P < 0.001) independent of other baseline characteristics. Aspirin 100 mg daily decreased U-TXM similarly across all tumour types (median reductions: 77-82%). Aspirin 300 mg daily provided no additional suppression of U-TXM compared with 100 mg. CONCLUSIONS: Persistently increased thromboxane biosynthesis was detected after radical cancer therapy, particularly in colorectal and gastro-oesophageal patients. Thromboxane biosynthesis should be explored further as a biomarker of active malignancy and may identify patients likely to benefit from aspirin.
Assuntos
Aspirina , Neoplasias Colorretais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores , Neoplasias Colorretais/tratamento farmacológico , Creatinina , Tromboxanos/uso terapêuticoRESUMO
At 125, aspirin still represents the cornerstone of anti-platelet therapy for the acute treatment and long-term prevention of atherothrombosis. The development of a selective regimen of low-dose aspirin for the inhibition of platelet thromboxane production was key to maximizing its antithrombotic efficacy and minimizing its gastrointestinal toxicity. Based on about 50 observational studies, published over the past 30 years, aspirin and other cyclooxygenase inhibitors have been associated with a reduced risk of colorectal cancer, and possibly other digestive tract cancers. The apparent chemopreventive effect of aspirin has been confirmed in post-hoc analyses of randomized cardiovascular trials and their meta-analyses. Moreover, prevention of sporadic colorectal adenoma recurrence was demonstrated by randomized controlled trials of low-dose aspirin and selective cyclooxygenase-2 inhibitors. A single placebo-controlled randomized trial of aspirin has shown long-term colorectal cancer prevention in patients with the Lynch syndrome. The sequential involvement of thromboxane-dependent platelet activation and cyclooxygenase-2-driven inflammatory response in the early stages of colorectal carcinogenesis may explain these clinical benefits. The aim of this mini-review is to analyze the existing evidence for a chemopreventive effect of aspirin and other cyclooxygenase inhibitors and discuss the missing pieces of this mechanistic and clinical puzzle. SIGNIFICANCE STATEMENT: Low-dose aspirin and other cyclooxygenase inhibitors have been associated with a reduced risk of colorectal cancer, and possibly other digestive tract cancers. The sequential involvement of thromboxane-dependent platelet activation and cyclooxygenase-2-driven inflammatory response in the early stages of colorectal carcinogenesis may explain these clinical benefits. The aim of this mini-review is to analyze the evidence for a chemopreventive effect of aspirin and other cyclooxygenase inhibitors and discuss the missing pieces of this mechanistic and clinical puzzle.
Assuntos
Neoplasias Colorretais , Neoplasias Gastrointestinais , Humanos , Ciclo-Oxigenase 2 , Aspirina/farmacologia , Aspirina/uso terapêutico , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/prevenção & controle , Neoplasias Gastrointestinais/tratamento farmacológico , Tromboxanos , Carcinogênese , Ciclo-Oxigenase 1 , Inibidores da Agregação Plaquetária , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
INTRODUCTION: The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting-at least partially-through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease. METHODS: After stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function. RESULTS: Our results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1-/-) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol. CONCLUSION: Amitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.
Assuntos
Asma , Broncoconstrição , Camundongos , Ratos , Humanos , Animais , Cobaias , Cloreto de Metacolina/farmacologia , Amitriptilina/farmacologia , Amitriptilina/uso terapêutico , Histamina/farmacologia , Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Serotonina/farmacologia , Serotonina/uso terapêutico , Acetilcolina/farmacologia , Simpatomiméticos/farmacologia , Simpatomiméticos/uso terapêutico , 1-Metil-3-Isobutilxantina/farmacologia , 1-Metil-3-Isobutilxantina/uso terapêutico , Dilatação , Pulmão , Asma/tratamento farmacológico , Albuterol , Endotelinas/farmacologia , Endotelinas/uso terapêutico , Tromboxanos/farmacologia , Tromboxanos/uso terapêuticoRESUMO
BACKGROUND: Surgical resection followed by indicated adjuvant therapy offers potential curative treatment in colonic adenocarcinoma. Beyond the well-established seed and soil theory of colon cancer progression, the 'normal-appearing' tissues near the tumor are not genuinely normal and remain as remnants in patients following surgery. Our objective was to elucidate the alteration of gene expression and pathways across various distances of resection margins in right-sided colonic adenocarcinoma. METHODS: Twenty-seven fresh samples of primary cancer and 56 matched non-tumor tissues adjacent to the tumor (NAT) were collected from patients with resectable right-sided colon cancer. NAT were systematically obtained at varying distances (1, 5, and 10 cm) on both proximal and distal sides. Comprehensive gene expression analysis was performed using 770-gene PanCancer Progression Panel, delineating distinctive pathways and functional predictions for each region. RESULTS: Distinctive gene signatures and pathways exhibited by normal-appearing tissues were discovered at varying distances from cancer. Notably, SFRP2, PTGDS, COL1A1, IL1B, THBS2, PTGIS, COL1A2, NPR1, and BGN were upregulated, while ENPEP, MMP1, and NRCAM were downregulated significantly in 1-cm tissue compared to farther distances. Substantial alterations in the extracellular matrix (ECM) and prostaglandin/thromboxane synthesis were significantly evident at the 1-cm distance. Functional analysis indicated enhanced cell viability and survival, alongside reduced cellular death and apoptosis. CONCLUSIONS: Different distances exerted a significant impact on gene alteration within the normal-looking mucosa surrounding primary cancer, influenced by various mechanisms. These findings may highlight potential therapeutic targets related to the ECM and prostaglandin/thromboxane pathways for treatment strategies.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Humanos , Prostaglandinas , Margens de Excisão , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Adenocarcinoma/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/cirurgia , Matriz Extracelular/genética , Matriz Extracelular/patologia , TromboxanosRESUMO
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.
Assuntos
Células Endoteliais , Receptores de Tromboxanos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/farmacologia , Células Endoteliais/metabolismo , Retroalimentação , Homeostase , Humanos , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Tromboxano A2/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacologiaRESUMO
BACKGROUND: Phospholipases A2 (PLA2 ) may be involved in α1 -adrenergic contraction by formation of thromboxane A2 in different smooth muscle types. However, whether this mechanism occurs with α1 -adrenergic contractions of the prostate, is still unknown. While α1 -adrenoceptor antagonists are the first line option for medical treatment of voiding symptoms in benign prostatic hyperplasia (BPH), improvements are limited, probably by nonadrenergic contractions including thromboxane A2 . Here, we examined effects of PLA2 inhibitors on contractions of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS) and by α1 -adrenergic agonists in an organ bath, after application of the cytosolic PLA2 inhibitors ASB14780 and AACOCF3, the secretory PLA2 inhibitor YM26734, the leukotriene receptor antagonist montelukast, or of solvent to controls. RESULTS: Frequency-dependent contractions of human prostate tissues induced by EFS were inhibited by 25% at 8 Hz, 38% at 16 Hz and 37% at 32 Hz by ASB14780 (1 µM), and by 32% at 16 Hz and 22% at 32 Hz by AACOCF3 (10 µM). None of both inhibitors affected contractions induced by noradrenaline, phenylephrine or methoxamine. YM26734 (3 µM) and montelukast (0.3 and 1 µM) neither affected EFS-induced contractions, nor contractions by α1 -adrenergic agonists, while all contractions were substantially inhibited by silodosin (100 nM). CONCLUSIONS: Our findings suggest presynaptic PLA2 functions in prostate smooth muscle contraction, while contractions induced by α1 -adrenergic agonists occur PLA2 -independent. Lacking sensitivity to montelukast excludes an involvement of PLA2 -derived leukotrienes in promotion of contractile neurotransmission.
Assuntos
Contração Muscular , Próstata , Masculino , Humanos , Próstata/fisiologia , Contração Muscular/fisiologia , Tromboxanos/farmacologia , Transmissão Sináptica , Agonistas Adrenérgicos/farmacologia , Músculo Liso , Adrenérgicos/farmacologia , Fosfolipases/farmacologiaRESUMO
BACKGROUND: Accumulating evidence suggests that prostaglandin E2, an arachidonic acid (AA) metabolite, enhances lymphangiogenesis in response to inflammation. However, thromboxane A2 (TXA2), another AA metabolite, is not well known. Thus, this study aimed to determine the role of thromboxane prostanoid (TP) signaling in lymphangiogenesis in secondary lymphedema. METHODS AND RESULTS: Lymphedema was induced by the ablation of lymphatic vessels in mouse tails. Compared with wild-type mice, tail lymphedema in Tp-deficient mice was enhanced, which was associated with suppressed lymphangiogenesis as indicated by decreased lymphatic vessel area and pro-lymphangiogenesis-stimulating factors. Numerous macrophages were found in the tail tissues of Tp-deficient mice. Furthermore, the deletion of TP in macrophages increased tail edema and decreased lymphangiogenesis and pro-lymphangiogenic cytokines, which was accompanied by increased numbers of macrophages and gene expression related to a pro-inflammatory macrophage phenotype in tail tissues. In vivo microscopic studies revealed fluorescent dye leakage in the lymphatic vessels in the wounded tissues. CONCLUSIONS: The results suggest that TP signaling in macrophages promotes lymphangiogenesis and prevents tail lymphedema. TP signaling may be a therapeutic target for improving lymphedema-related symptoms by enhancing lymphangiogenesis.
Assuntos
Vasos Linfáticos , Linfedema , Camundongos , Animais , Linfangiogênese , Prostaglandinas/metabolismo , Tromboxanos/metabolismo , Vasos Linfáticos/metabolismo , Macrófagos/metabolismo , Linfedema/genética , Linfedema/metabolismoRESUMO
Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.
Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase , Inibidores de Lipoxigenase , Humanos , Inibidores de Lipoxigenase/farmacologia , Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Solubilidade , Leucócitos Mononucleares/metabolismo , Epóxido Hidrolases/metabolismo , Inibidores Enzimáticos/farmacologia , Anti-Inflamatórios/farmacologia , Piridinas/farmacologia , Tromboxanos , LipídeosRESUMO
BACKGROUND: Pelacarsen decreases plasma levels of lipoprotein(a) [Lp(a)] and oxidized phospholipids (OxPL). It was previously reported that pelacarsen does not affect the platelet count. We now report the effect of pelacarsen on on-treatment platelet reactivity. METHODS: Subjects with established cardiovascular disease and screening Lp(a) levels ≥60 mg per deciliter (~ ≥150 nmol/L) were randomized to receive pelacarsen (20, 40, or 60 mg every 4 weeks; 20 mg every 2 weeks; or 20 mg every week), or placebo for 6-12 months. Aspirin Reaction Units (ARU) and P2Y12 Reaction Units (PRU) were measured at baseline and the primary analysis timepoint (PAT) at 6 months. RESULTS: Of the 286 subjects randomized, 275 had either an ARU or PRU test, 159 (57.8%) were on aspirin alone and 94 (34.2%) subjects were on dual anti-platelet therapy. As expected, the baseline ARU and PRU were suppressed in subjects on aspirin or on dual anti-platelet therapy, respectively. There were no significant differences in baseline ARU in the aspirin groups or in PRU in the dual anti-platelet groups. At the PAT there were no statistically significant differences in ARU in subjects on aspirin or PRU in subjects on dual anti-platelet therapy among any of the pelacarsen groups compared to the pooled placebo group (p > 0.05 for all comparisons). CONCLUSION: Pelacarsen does not modify on-treatment platelet reactivity through the thromboxane A2 or P2Y12 platelet receptor pathways.
Assuntos
Inibidores da Agregação Plaquetária , Tromboxanos , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Clopidogrel/farmacologia , Estudos Prospectivos , Plaquetas , Aspirina/uso terapêutico , Testes de Função Plaquetária , Resultado do Tratamento , Antagonistas do Receptor Purinérgico P2Y/uso terapêuticoRESUMO
Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-ß (transforming growth factor-ß) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Receptores de Tromboxanos , Animais , Bleomicina/farmacologia , F2-Isoprostanos/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/genética , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Prostaglandinas/metabolismo , Receptores de Tromboxanos/metabolismo , Tromboxanos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.
Assuntos
Trombina , Tromboxanos , Animais , Camundongos , Plaquetas , Fibrinolíticos/farmacologia , Agregação Plaquetária , Trombina/farmacologia , Tromboxano A2 , Umbeliferonas/farmacologia , Fosfolipases A2 Citosólicas/metabolismoRESUMO
Platelets play crucial roles in cardiovascular diseases (CVDs) by regulating hemostasis and blood coagulation at sites of blood vessel damage. Accumulating evidence indicates daidzein inhibits platelet activation, but the mechanism involved has not been elucidated. Thus, in this study, we investigated the mechanism responsible for the inhibition of collagen-induced platelet aggregation by daidzein. We found that in collagen-induced platelets, daidzein suppressed the production of thromboxane A2 (TXA2), a molecule involved in platelet activation and aggregation, by inhibiting the cytosolic phospholipase A2 (cPLA2) signaling pathway. However, daidzein did not affect cyclooxygenase-1 (COX-1). Furthermore, daidzein attenuated the PI3K/PDK1/Akt/GSK3αß and MAPK (p38, ERK) signaling pathways, increased the phosphorylation of inositol trisphosphate receptor1 (IP3R1) and vasodilator-stimulated phosphoprotein (VASP), and increased the level of cyclic adenosine monophosphate (cAMP). These results suggest that daidzein inhibits granule release (ATP, serotonin, P-selectin), integrin αIIbß3 activation, and clot retraction. Taken together, our study demonstrates that daidzein inhibits collagen-induced platelet aggregation and suggests that daidzein has therapeutic potential for the treatment of platelet aggregation-related diseases such as atherosclerosis and thrombosis.
Assuntos
Ativação Plaquetária , Inibidores da Agregação Plaquetária , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária , Plaquetas/metabolismo , Fosforilação , Tromboxanos/metabolismo , Colágeno/metabolismoRESUMO
Introduction: Pulmonary hypertension due to chronic obstructive pulmonary disease (COPD) is classified as Group 3 pulmonary hypertension, with no current proven targeted therapies. It has been shown that cigarette smoke, the main risk factor for COPD, can increase thromboxane A2 production in healthy human pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, and that blocking the effect of increased thromboxane A2 using daltroban, a thromboxane A2 receptor antagonist, can inhibit cigarette smoke-induced pulmonary artery cell proliferation. However, it is largely unknown whether thromboxane A2 is increased in smokers with COPD. Therefore, the aim of this study was to assess the level of thromboxane A2 production in patients with COPD who smoke. Methods: Pulmonary artery smooth muscle cells from three smokers with COPD and three healthy donors were cultured in cell culture medium. The culture medium was collected and the thromboxane B2 (a stable metabolite of thromboxane A2) released in the culture medium was quantified using an ELISA kit. The data were normalised with the total protein concentration and then expressed in pg/mg protein. Demographic data were collected and baseline pulmonary function tests of patients with COPD were conducted. Results: The mean age of patients with COPD was 69 ± 7 years. All patients were smokers and had a mean smoking history of 39.66 ± 9.50 packs per year. The mean forced expiratory volume in one second, that is, FEV1%, and the ratio of forced vital capacity (FVC) to FEV1% of COPD patients were 63.33 ± 19.60% and 52.66 ± 14.64%, respectively. The results revealed that thromboxane A2 production was significantly increased in pulmonary artery smooth muscle cells from smokers with COPD (434.56 ± 82.88 pg/mg protein) compared with the thromboxane A2 levels in pulmonary artery smooth muscle cells from healthy donors (160 ± 59.3 pg/mg protein). Conclusions: This is the first report of increased thromboxane A2 production in pulmonary artery smooth muscle cells from smokers with COPD. This observation strongly suggests that thromboxane A2 can be used as a novel therapeutic target for the treatment of patients with COPD-associated pulmonary hypertension.
Assuntos
Hipertensão Pulmonar , Doença Pulmonar Obstrutiva Crônica , Humanos , Pessoa de Meia-Idade , Idoso , Artéria Pulmonar , Tromboxanos , Células Endoteliais , Doença Pulmonar Obstrutiva Crônica/complicações , Miócitos de Músculo LisoRESUMO
Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.