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Coffee is one of the most widely consumed beverages in the world due to its unique aroma and psychostimulant effects, mainly due to the presence of caffeine. In recent years, experimental evidence has shown that the moderate consumption of coffee (3/4 cups per day) is safe and beneficial to human health, revealing protective effects against numerous chronic metabolic diseases such as diabetes, cardiovascular, neurodegenerative, and hepatic diseases. This review focuses on two of coffee's main bioactive compounds, i.e., caffeine and chlorogenic acids, and their effects on the progression of chronic liver diseases, demonstrating that regular coffee consumption correlates with a lower risk of the development and progression of non-alcoholic steatohepatitis, viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. In particular, this review analyzes caffeine and chlorogenic acid from a pharmacological point of view and explores the molecular mechanism through which these compounds are responsible for the protective role of coffee. Both bioactive compounds, therefore, have antifibrotic effects on hepatic stellate cells and hepatocytes, induce a decrease in connective tissue growth factor, stimulate increased apoptosis with anti-cancer effects, and promote a major inhibition of focal adhesion kinase, actin, and protocollagen synthesis. In conclusion, coffee shows many beneficial effects, and experimental data in favor of coffee consumption in patients with liver diseases are encouraging, but further prospective studies are needed to demonstrate its preventive and therapeutic role in chronic liver diseases.
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New insights have been gained on the role of platelets beyond thrombosis. Platelets can accumulate in damaged and inflamed tissues, acting as a sentinel to detect and repair tissue damage. However, by releasing several soluble factors, including thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid, and extracellular vesicles (EVs), platelets can activate vascular cells, stromal, such as fibroblasts, immune cells, and cancer cells, leading to atherosclerosis, vascular restenosis, tissue fibrosis, and tumor metastasis. Platelet-derived extracellular vesicles (PEVs) are released when platelets are activated and can transfer their cargo to other cell types, thus contributing to the development of diseases. Inhibitors of the internalization of PEVs can potentially represent novel therapeutic tools. Both platelets and PEVs contain a significant number of different types of molecules, and their omics assessment and integration with clinical data using computational approaches have the potential to detect early disease development and monitor drug treatments.
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Plaquetas , Eicosanoides , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Plaquetas/metabolismo , Plaquetas/patologia , Eicosanoides/metabolismo , Animais , Comunicação CelularRESUMO
Background: Low-dose aspirin's mechanism of action for preventing colorectal cancer (CRC) is still debated, and the optimal dose remains uncertain. We aimed to optimize the aspirin dose for cancer prevention in CRC patients through deep phenotyping using innovative biomarkers for aspirin's action. Methods: We conducted a Phase II, open-label clinical trial in 34 CRC patients of both sexes randomized to receive enteric-coated aspirin 100 mg/d, 100 mg/BID, or 300 mg/d for 3 ± 1 weeks. Biomarkers were evaluated in blood, urine, and colorectal biopsies at baseline and after dosing with aspirin. Novel biomarkers of aspirin action were assessed in platelets and colorectal tissues using liquid chromatography-mass spectrometry to quantify the extent of cyclooxygenase (COX)-1 and COX-2 acetylation at Serine 529 and Serine 516, respectively. Results: All aspirin doses caused comparable % acetylation of platelet COX-1 at Serine 529 associated with similar profound inhibition of platelet-dependent thromboxane (TX)A2 generation ex vivo (serum TXB2) and in vivo (urinary TXM). TXB2 was significantly reduced in CRC tissue by aspirin 300 mg/d and 100 mg/BID, associated with comparable % acetylation of COX-1. Differently, 100 mg/day showed a lower % acetylation of COX-1 in CRC tissue and no significant reduction of TXB2. Prostaglandin (PG)E2 biosynthesis in colorectal tumors and in vivo (urinary PGEM) remained unaffected by any dose of aspirin associated with the variable and low extent of COX-2 acetylation at Serine 516 in tumor tissue. Increased expression of tumor-promoting genes like VIM (vimentin) and TWIST1 (Twist Family BHLH Transcription Factor 1) vs. baseline was detected with 100 mg/d of aspirin but not with the other two higher doses. Conclusion: In CRC patients, aspirin 300 mg/d or 100 mg/BID had comparable antiplatelet effects to aspirin 100 mg/d, indicating similar inhibition of the platelet's contribution to cancer. However, aspirin 300 mg/d and 100 mg/BID can have additional anticancer effects by inhibiting cancerous tissue's TXA2 biosynthesis associated with a restraining impact on tumor-promoting gene expression. EUDRACT number: 2018-002101-65. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03957902.
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Cyclooxygenase (COX) isozymes, i.e., COX-1 and COX-2, are encoded by separate genes and are involved in the generation of the same products, prostaglandin (PG)G2 and PGH2 from arachidonic acid (AA) by the COX and peroxidase activities of the enzymes, respectively. PGH2 is then transformed into prostanoids in a tissue-dependent fashion due to the different expression of downstream synthases. Platelets present almost exclusively COX-1, which generates large amounts of thromboxane (TX)A2, a proaggregatory and vasoconstrictor mediator. This prostanoid plays a central role in atherothrombosis, as shown by the benefit of the antiplatelet agent low-dose aspirin, a preferential inhibitor of platelet COX-1. Recent findings have shown the relevant role played by platelets and TXA2 in developing chronic inflammation associated with several diseases, including tissue fibrosis and cancer. COX-2 is induced in response to inflammatory and mitogenic stimuli to generate PGE2 and PGI2 (prostacyclin), in inflammatory cells. However, PGI2 is constitutively expressed in vascular cells in vivo and plays a crucial role in protecting the cardiovascular systems due to its antiplatelet and vasodilator effects. Here, platelets' role in regulating COX-2 expression in cells of the inflammatory microenvironment is described. Thus, the selective inhibition of platelet COX-1-dependent TXA2 by low-dose aspirin prevents COX-2 induction in stromal cells leading to antifibrotic and antitumor effects. The biosynthesis and functions of other prostanoids, such as PGD2, and isoprostanes, are reported. In addition to aspirin, which inhibits platelet COX-1 activity, possible strategies to affect platelet functions by influencing platelet prostanoid receptors or synthases are discussed.
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Aspirina , Prostaglandinas , Humanos , Ciclo-Oxigenase 2 , Aspirina/farmacologia , Aspirina/uso terapêutico , Tromboxano A2/fisiologia , Prostaglandina H2RESUMO
BACKGROUND: The results of Aspirin prevention of colorectal adenomas in patients with familial adenomatous polyposis (FAP) are controversial. METHODS: We conducted a biomarker-based clinical study in eight FAP patients treated with enteric-coated low-dose Aspirin (100 mg daily for three months) to explore whether the drug targets mainly platelet cyclooxygenase (COX)-1 or affects extraplatelet cellular sources expressing COX-isozymes and/or off-target effects in colorectal adenomas. RESULTS: In FAP patients, low-dose Aspirin-acetylated platelet COX-1 at Serine529 (>70%) was associated with an almost complete inhibition of platelet thromboxane (TX) B2 generation ex vivo (serum TXB2). However, enhanced residual urinary 11-dehydro-TXB2 and urinary PGEM, primary metabolites of TXA2 and prostaglandin (PG)E2, respectively, were detected in association with incomplete acetylation of COX-1 in normal colorectal biopsies and adenomas. Proteomics of adenomas showed that Aspirin significantly modulated only eight proteins. The upregulation of vimentin and downregulation of HBB (hemoglobin subunit beta) distinguished two groups with high vs. low residual 11-dehydro-TXB2 levels, possibly identifying the nonresponders and responders to Aspirin. CONCLUSIONS: Although low-dose Aspirin appropriately inhibited the platelet, persistently high systemic TXA2 and PGE2 biosynthesis were found, plausibly for a marginal inhibitory effect on prostanoid biosynthesis in the colorectum. Novel chemotherapeutic strategies in FAP can involve blocking the effects of TXA2 and PGE2 signaling with receptor antagonists.
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BACKGROUND: Platelet-cancer cell interactions modulate tumor metastasis and thrombosis in cancer. Platelet-derived extracellular vesicles (EVs) can contribute to these outcomes. METHODS: We characterized the medium-sized EVs (mEVs) released by thrombin-stimulated platelets of colorectal cancer (CRC) patients and healthy subjects (HS) on the capacity to induce epithelial-mesenchymal transition (EMT)-related genes and cyclooxygenase (COX)-2(PTGS2), and thromboxane (TX)B2 production in cocultures with four colorectal cancer cell lines. Platelet-derived mEVs were assessed for their size distribution and proteomics signature. RESULTS: The mEV population released from thrombin-activated platelets of CRC patients had a different size distribution vs. HS. Platelet-derived mEVs from CRC patients, but not from HS, upregulated EMT marker genes, such as TWIST1 and VIM, and downregulated CDH1. PTGS2 was also upregulated. In cocultures of platelet-derived mEVs with cancer cells, TXB2 generation was enhanced. The proteomics profile of mEVs released from activated platelets of CRC patients revealed that 119 proteins were downregulated and 89 upregulated vs. HS. CONCLUSIONS: We show that mEVs released from thrombin-activated platelets of CRC patients have distinct features (size distribution and proteomics cargo) vs. HS and promote prometastatic and prothrombotic phenotypes in cancer cells. The analysis of platelet-derived mEVs from CRC patients could provide valuable information for developing an appropriate treatment plan.
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Clinical and experimental evidence sustain the role of cyclooxygenase (COX)-1 in intestinal tumorigenesis. However, the cell type expressing the enzyme involved and molecular mechanism(s) have not been clarified yet. We aimed to elucidate the role of platelet COX-1 (the target of low-dose aspirin in humans) in intestinal tumorigenesis of ApcMin/+ mice, considered a clinically relevant model. To realize this objective, we generated an ApcMin/+ mouse with a specific deletion of Ptgs1(COX-1 gene name) in megakaryocytes/platelets (ApcMin/+;pPtgs1-/-mice) characterized by profound inhibition of thromboxane(TX)A2 biosynthesis ex vivo (serum TXB2; by 99%) and in vivo [urinary 2,3-dinor-TXB2(TXM), by 79%]. ApcMin/+ mice with the deletion of platelet COX-1 showed a significantly reduced number (67%) and size (32%) of tumors in the small intestine. The intestinal adenomas of these mice had decreased proliferative index associated with reduced COX-2 expression and systemic prostaglandin(PG)E2 biosynthesis (urinary PGEM) vs. ApcMin/+mice. Extravasated platelets were detected in the intestine of ApcMin/+mice. Thus, we explored their contribution to COX-2 induction in fibroblasts, considered the primary polyp cell type expressing the protein. In the coculture of human platelets and myofibroblasts, platelet-derived TXA2 was involved in the induction of COX-2-dependent PGE2 in myofibroblasts since it was prevented by the selective inhibition of platelet COX-1 by aspirin or by a specific antagonist of TXA2 receptors. In conclusion, our results support the platelet hypothesis of intestinal tumorigenesis and provide experimental evidence that selective inhibition of platelet COX-1 can mitigate early events of intestinal tumorigenesis by restraining COX-2 induction.
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Polipose Intestinal , Megacariócitos , Camundongos , Humanos , Animais , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Transformação Celular Neoplásica , Carcinogênese , Aspirina/farmacologiaRESUMO
Platelet-type lipoxygenase (pl12-LOX), encoded by ALOX12, catalyzes the production of the lipid mediator 12S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12S-HpETE), which is quickly reduced by cellular peroxidases to form 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12S-HETE). Platelets express high levels of pl12-LOX and generate considerable amounts of 12S-HETE from arachidonic acid (AA; C20:4, n-6). The development of sensitive chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods has allowed the accurate quantification of 12S-HETE in biological samples. Moreover, advances in the knowledge of the mechanism of action of 12S-HETE have been achieved. The orphan G-protein-coupled receptor 31 (GPR31) has been identified as the high-affinity 12S-HETE receptor. Moreover, upon platelet activation, 12S-HETE is produced, and significant amounts are found esterified to membrane phospholipids (PLs), such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), promoting thrombin generation. Platelets play many roles in cancer metastasis. Among them, the platelets' ability to interact with cancer cells and transfer platelet molecules by the release of extracellular vesicles (EVs) is noteworthy. Recently, it was found that platelets induce epithelial-mesenchymal transition(EMT) in cancer cells, a phenomenon known to confer high-grade malignancy, through the transfer of pl12-LOX contained in platelet-derived EVs. These cancer cells now generate 12-HETE, considered a key modulator of cancer metastasis. Interestingly, 12-HETE was mainly found esterified in plasmalogen phospholipids of cancer cells. This review summarizes the current knowledge on the regulation and functions of pl12-LOX in platelets and cancer cells and their crosstalk.Novel approaches to preventing cancer and metastasis by the pharmacological inhibition of pl12-LOX and the internalization of mEVs are discussed.
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Araquidonato 12-Lipoxigenase , Neoplasias , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Fosfatidiletanolaminas , Ácido Araquidônico , Trombina , Plasmalogênios , Cromatografia Líquida , Espectrometria de Massas em Tandem , Plaquetas , Fosfatidilcolinas , Biologia , Peroxidases , Ácidos HidroxieicosatetraenoicosRESUMO
Colorectal (CRC) and hepatocellular carcinoma (HCC) are associated with chronic inflammation, which plays a role in tumor development and malignant progression. An unmet medical need in these settings is the availability of sensitive and specific noninvasive biomarkers. Their use will allow surveillance of high-risk populations, early detection, and monitoring of disease progression. Moreover, the characterization of specific fingerprints of patients with nonalcoholic fatty liver disease (NAFLD) without or with nonalcoholic steatohepatitis (NASH) at the early stages of liver fibrosis is necessary. Some lines of evidence show the contribution of platelets to intestinal and liver inflammation. Thus, low-dose Aspirin, an antiplatelet agent, reduces CRC and liver cancer incidence and mortality. Aspirin also produces antifibrotic effects in NAFLD. Activated platelets can trigger chronic inflammation and tissue fibrosis via the release of soluble mediators, such as thromboxane (TX) A2 and tumor growth factor (TGF)-ß, and vesicles containing genetic material (including microRNA). These platelet-derived products contribute to cyclooxygenase (COX)-2 expression and prostaglandin (PG)E2 biosynthesis by tumor microenvironment cells, such as immune and endothelial cells and fibroblasts, alongside cancer cells. Enhanced COX-2-dependent PGE2 plays a crucial role in chronic inflammation and promotes tumor progression, angiogenesis, and metastasis. Antiplatelet agents can indirectly prevent the induction of COX-2 in target cells by inhibiting platelet activation. Differently, selective COX-2 inhibitors (coxibs) block the activity of COX-2 expressed in the tumor microenvironment and cancer cells. However, coxib chemopreventive effects are hampered by the interference with cardiovascular homeostasis via the coincident inhibition of vascular COX-2-dependent prostacyclin biosynthesis, resulting in enhanced risk of atherothrombosis. A strategy to improve anti-inflammatory agents' use in cancer prevention could be to develop tissue-specific drug delivery systems. Platelet ability to interact with tumor cells and transfer their molecular cargo can be employed to design platelet-mediated drug delivery systems to enhance the efficacy and reduce toxicity associated with anti-inflammatory agents in these settings. Another peculiarity of platelets is their capability to uptake proteins and transcripts from the circulation. Thus, cancer patient platelets show specific proteomic and transcriptomic expression profiles that could be used as biomarkers for early cancer detection and disease monitoring.
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Metastasis requires that cancer cells survive in the circulation, colonize distant organs, and grow. Despite platelets being central contributors to hemostasis, leukocyte trafficking during inflammation, and vessel stability maintenance, there is significant evidence to support their essential role in supporting metastasis through different mechanisms. In addition to their direct interaction with cancer cells, thus forming heteroaggregates such as leukocytes, platelets release molecules that are necessary to promote a disseminating phenotype in cancer cells via the induction of an epithelial-mesenchymal-like transition. Therefore, agents that affect platelet activation can potentially restrain these prometastatic mechanisms. Although the primary adhesion of platelets to cancer cells is mainly independent of G protein-mediated signaling, soluble mediators released from platelets, such as ADP, thromboxane (TX) A2, and prostaglandin (PG) E2, act through G protein-coupled receptors (GPCRs) to cause the activation of more additional platelets and drive metastatic signaling pathways in cancer cells. In this review, we examine the contribution of the GPCRs of platelets and cancer cells in the development of cancer metastasis. Finally, the possible use of agents affecting GPCR signaling pathways as antimetastatic agents is discussed.
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Neoplasias , Inibidores da Agregação Plaquetária , Plaquetas/metabolismo , Humanos , Neoplasias/metabolismo , Ativação Plaquetária , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Receptores Acoplados a Proteínas G/metabolismo , Tromboxano A2/metabolismo , Tromboxano A2/farmacologiaRESUMO
Background: Aspirin(acetylsalicylic acid, ASA) is recommended for the secondary prevention of atherothrombotic events and has shown anticancer effects. The current enteric-coated drug formulation may reduce aspirin bioavailability. Liquid formulations could improve aspirin pharmacokinetics and pharmacodynamics. IP1867B is a liquid-aspirin formulation that combines three ingredients, ASA/triacetin/saccharin. Methods: ASA and IP1867B(L-ASA) were assessed in human serum(obtained by allowing to clot human whole blood at 37 °C for 1h), washed platelets, and colonic adenocarcinoma HCA7 cells on eicosanoid generation and COX-isozyme acetylation at Serine529 and 516 by LC-MS/MS. Results: In serum, ASA and L-ASA acted by selectively affecting COX-1-derived eicosanoids, including thromboxane(TX)B2. L-ASA was more potent in inhibiting serum TXB2, a known biomarker of aspirin antiplatelet effect, than ASA. However, ASA and L-ASA were equipotent to acetylate COX-1 in washed platelets and COX-2 in HCA7 cells. In HCA7 cells, ASA and L-ASA acted by inhibiting prostaglandin(PG)E2(the most abundant prostanoid) and TXB2 biosynthesis. In the presence of a high arachidonic acid concentration(100 µM), 15R-hydroxyeicosatetraenoic acid(HETE) was generated at baseline by cancer cell COX-2 and was only slightly enhanced by supratherapeutic concentrations of ASA(1 mM). In whole blood and HCA7 cells treated with ASA or L-ASA, 15-epi-lipoxin(LX)A4 were undetectable. Conclusion: IP1867B was more potent in affecting serum TXB2 generation than ASA. The relevance of this finding deserves evaluation in vivo in humans. In cancer cells, ASA and IP1867B acted by inhibiting PGE2 and TXB2 generation via the acetylation of COX-2. ASA and IP867B at clinically relevant concentrations did not substantially induce the biosynthesis of 15R-HETE and 15-epi-LXA4.
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Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Araquidonato 12-Lipoxigenase/metabolismo , Plaquetas/metabolismo , Neoplasias do Colo/metabolismo , Fosfolipídeos/metabolismo , Adulto , Neoplasias do Colo/patologia , Humanos , Pessoa de Meia-Idade , Células Tumorais Cultivadas , Adulto JovemRESUMO
Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A2 in their pathophysiology remains unclear. We investigated the systemic TXA2 biosynthesis in vivo and gene expression of its receptor TP in 22 essential hypertension patients and a mouse model of salt-sensitive hypertension. The contribution of platelet TXA2 biosynthesis on enhanced blood pressure (BP) and overload-induced cardiac fibrosis was explored in mice by treating with low-dose Aspirin, resulting in selective inhibition of platelet cyclooxygenase (COX)-1-dependent TXA2 generation. In essential hypertensive patients, systemic biosynthesis of TXA2 [assessed by measuring its urinary metabolites (TXM) reflecting predominant platelet source] was enhanced together with higher gene expression of circulating leukocyte TP and TGF-ß, vs. normotensive controls. Similarly, in hypertensive mice with prostacyclin (PGI2) receptor (IP) deletion (IPKO) fed with a high-salt diet, enhanced urinary TXM, and left ventricular TP overexpression were detected vs. normotensive wildtype (WT) mice. Increased cardiac collagen deposition and profibrotic gene expression (including TGF-ß) was found. Low-dose Aspirin administration caused a selective inhibition of platelet TXA2 biosynthesis and mitigated enhanced blood pressure, cardiac fibrosis, and left ventricular profibrotic gene expression in IPKO but not WT mice. Moreover, the number of myofibroblasts and extravasated platelets in the heart was reduced. In cocultures of human platelets and myofibroblasts, platelet TXA2 induced profibrotic gene expression, including TGF-ß1. In conclusion, our results support tailoring low-dose Aspirin treatment in hypertensive patients with unconstrained TXA2/TP pathway to reduce blood pressure and prevent early cardiac fibrosis.
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Antifibróticos/farmacologia , Anti-Hipertensivos/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Cardiomiopatias/prevenção & controle , Hipertensão Essencial/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Tromboxano A2/sangue , Adulto , Animais , Biomarcadores/sangue , Plaquetas/metabolismo , Cardiomiopatias/sangue , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Hipertensão Essencial/sangue , Hipertensão Essencial/complicações , Hipertensão Essencial/fisiopatologia , Feminino , Fibrose , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Receptores de Epoprostenol/genética , Receptores de Epoprostenol/metabolismo , Receptores de Tromboxanos/metabolismoRESUMO
BACKGROUND: Ion channels are transmembrane proteins that play important roles in cell function regulation modulating ionic cell permeability. In megakaryocytes and platelets, regulated ion flows have been demonstrated to modulate platelet production and function. However, a relatively limited characterization of ion channel expression and function is available in the human megakaryocyte-platelet lineage. OBJECTIVE: We analyzed the expression and function of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 (also known as Maxi-K, BK, slo1) in human megakaryocytes and platelets. METHODS: To investigate the functionality of Kca 1.1, we exploited different agonists (BMS-191011, NS1619, NS11021, epoxyeicosatrienoic acid isoforms) and inhibitors (iberiotoxin, penitrem A) of the channel. RESULTS: In megakaryocytes, Kca 1.1 agonists determined a decreased proplatelet formation and altered interaction with the extracellular matrix. Analysis of the actin cytoskeleton demonstrated a significant decrease in megakaryocyte spreading and adhesion to collagen. In platelets, the opening of the channel Kca 1.1 led to a reduced sensitivity to agonists with blunted aggregation in response to ADP, with an inhibitory capacity additive to that of aspirin. The Kca 1.1 agonists, but not the inhibitors, determined a reduction of platelet adhesion and aggregation onto immobilized collagen underflow to an extent similar to that of aspirin and ticagrelor. The opening of the Kca 1.1 resulted in cell hyperpolarization impairing free intracellular calcium in ADP-stimulated platelets and megakaryocytes. CONCLUSIONS: The present study reveals new mechanisms in platelet formation and activation, suggesting that targeting Kca 1.1 channels might be of potential pharmacological interest in hemostasis and thrombosis.
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Cálcio , Megacariócitos , Benzimidazóis , Plaquetas , Humanos , Canais de PotássioRESUMO
Neointima hyperplasia is a crucial component of restenosis after coronary angioplasty. We have hypothesized that enhanced generation of platelet-derived thromboxane (TX)A2 in response to vascular damage plays a critical role in neointimal hyperplasia and that antiplatelet agents may mitigate it. In cocultures of human platelets and coronary artery smooth muscle cells (CASMC), we found that platelets induced morphologic changes and enhanced the migration of CASMC. The exposure of platelets to Aspirin [an inhibitor of cyclooxygenase (COX)-1] reduced the generation of TXA2 and prevented the morphological and functional changes induced by platelets in CASMC. Platelet-derived TXA2 induced COX-2 and enhanced prostaglandin (PG)E2 biosynthesis in CASMC, a known mechanism promoting neointimal hyperplasia. COX-2 induction was prevented by different antiplatelet agents, i.e., Aspirin, the TP antagonist SQ29,548, or Revacept (a dimeric soluble GPVI-Fc fusion protein). The administration of the novel antiplatelet agent Revacept to C57BL/6 mice, beginning three days before femoral artery denudation, and continuing up to seven days after injury, prevented the increase of the systemic biosynthesis di TXA2 and reduced femoral artery intima-to-media area and the levels of markers of cell proliferation and macrophage infiltration. Revacept might serve as a therapeutic agent for percutaneous coronary angioplasty and stent implantation.
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Plaquetas/citologia , Vasos Coronários/citologia , Glicoproteínas/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Neointima/prevenção & controle , Inibidores da Agregação Plaquetária/farmacologia , Tromboxano A2/biossíntese , Urina/química , Adulto , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Ciclo-Oxigenase 2/metabolismo , Humanos , Hiperplasia , Masculino , Camundongos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima/metabolismo , Neointima/patologia , Adulto JovemRESUMO
Platelets contribute to several types of cancer through plenty of mechanisms. Upon activation, platelets release many molecules, including growth and angiogenic factors, lipids, and extracellular vesicles, and activate numerous cell types, including vascular and immune cells, fibroblasts, and cancer cells. Hence, platelets are a crucial component of cell-cell communication. In particular, their interaction with cancer cells can enhance their malignancy and facilitate the invasion and colonization of distant organs. These findings suggest the use of antiplatelet agents to restrain cancer development and progression. Another peculiarity of platelets is their capability to uptake proteins and transcripts from the circulation. Thus, cancer-patient platelets show specific proteomic and transcriptomic expression patterns, a phenomenon called tumor-educated platelets (TEP). The transcriptomic/proteomic profile of platelets can provide information for the early detection of cancer and disease monitoring. Platelet ability to interact with tumor cells and transfer their molecular cargo has been exploited to design platelet-mediated drug delivery systems to enhance the efficacy and reduce toxicity often associated with traditional chemotherapy. Platelets are extraordinary cells with many functions whose exploitation will improve cancer diagnosis and treatment.
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Plaquetas/metabolismo , Neoplasias/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Carcinogênese/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Biópsia Líquida/métodos , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B2 production; ASA selectively affected TXB2 production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB2 while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y12 blocker) affected platelet aggregation associated with profound inhibition of TXB2 generation. P2Y12 receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Prostaglandinas/metabolismo , Adulto , Aspirina/farmacologia , Plaquetas/metabolismo , Descoberta de Drogas , Humanos , Ácidos Hidroxieicosatetraenoicos/sangue , Lipidômica , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Prostaglandinas/sangue , Ticagrelor/farmacologia , Adulto JovemRESUMO
The most recognized mechanism of aspirin (acetylsalicylic acid, ASA) action, at therapeutic dosing, is the inhibition of prostanoid biosynthesis through the acetylation of cyclooxygenase (COX)-isozymes (COX-1 at serine-529 and COX-2 at serine-516). Whether aspirin, also when given at the low-doses recommended for cardiovascular prevention, reduces the risk of colorectal cancer by affecting COX-2 activity in colorectal adenomatous lesions is still debated. We aimed to develop a direct biomarker of aspirin action on COX-2 by assessing the extent of acetylation of COX-2 at serine-516 using the AQUA strategy, enabling absolute protein quantitation by liquid chromatography-mass spectrometry. We compared the extent of acetylation and the inhibition of prostanoid biosynthesis by ASA using human recombinant COX-2 (hu-COX-2), the human colon cancer cell line HCA-7, isolated human monocytes stimulated with LPS (lipopolysaccharide) or human intestinal epithelial cells stimulated with interleukin (IL)-1ß. Hu-COX-2 exposed in vitro to an excess of ASA was acetylated by approximately 40-50% associated with the inhibition of COX-2 activity by 80-90%. In the three cell-types expressing COX-2, the extent of COX-2 acetylation and reduction of prostaglandin (PG) E2 biosynthesis by ASA was concentration-dependent with comparable EC50 values (in the low µM range). The maximal % acetylation of COX-2 averaged 80%, at ASA 1000 µM, and was associated with a virtually complete reduction of PGE2 biosynthesis (97%). In conclusion, we have developed a proteomic assay to evaluate the extent of acetylation of COX-2 at serine-516 by aspirin; its use in clinical studies will allow clarifying the mechanism of action of aspirin as anticancer agent.
Assuntos
Aspirina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Acetilação , Sequência de Aminoácidos , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Espectrometria de Massas , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismoRESUMO
RATIONALE: The development of inhibitors of microsomal prostaglandin (PG)E2 synthase-1 (mPGES-1) was driven by the promise of attaining antiinflammatory agents with a safe cardiovascular profile because of the possible diversion of the accumulated substrate, PGH2, towards prostacyclin (PGI2). OBJECTIVES: We studied the effect of the human mPGES-1 inhibitor, AF3485 (a benzamide derivative) on prostanoid biosynthesis in human whole blood in vitro. To characterize possible off-target effects of the compound, we evaluated: i)the impact of its administration on the systemic biosynthesis of prostanoids in a model of complete Freund's adjuvant (CFA)-induced monoarthritis in rats; ii) the effects on cyclooxygenase (COX)-2 expression and the biosynthesis of prostanoids in human monocytes and human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Prostanoids were assessed in different cellular models by immunoassays. The effect of the administration of AF3485 (30 and 100 mg/kg,i.p.) or celecoxib (20mg/kg, i.p.), for 3 days, on the urinary levels of enzymatic metabolites of prostanoids, PGE-M, PGI-M, and TX-M were assessed by LC-MS. RESULTS: In LPS-stimulated whole blood, AF3485 inhibited PGE2 biosynthesis, in a concentration-dependent fashion. At 100µM, PGE2 levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB2 inhibition (40.56 ± 5.77%). AF3485 administration to CFA-treated rats significantly reduced PGE-M (P < 0.01) and TX-M (P < 0.05) similar to the selective COX-2 inhibitor, celecoxib. In contrast, AF3485 induced a significant (P < 0.05) increase of urinary PGI-M while it was reduced by celecoxib. In LPS-stimulated human monocytes, AF3485 inhibited PGE2 biosynthesis with an IC50 value of 3.03 µM (95% CI:0.5-8.75). At 1µM, AF3485 enhanced TXB2 while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB2. At 100 µM, maximal inhibition of the two prostanoids was associated with the downregulation of COX-2 protein by 86%. These effects did not involve AMPK pathway activation, IkB stabilization, or PPARγ activation. In HUVEC, AF3485 at 100 µM caused a significant (P < 0.05) induction of COX-2 protein associated with enhanced PGI2 production. These effects were reversed by the PPARγ antagonist GW9662. CONCLUSIONS: The inhibitor of human mPGES-1 AF3485 is a novel antiinflammatory compound which can also modulate COX-2 induction by inflammatory stimuli. The compound also induces endothelial COX-2-dependent PGI2 production via PPARγ activation, both in vitro and in vivo, which might translate into a protective effect for the cardiovascular system.
RESUMO
Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A2 production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA2 enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA2 receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA2 in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A2 in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD.