RESUMEN
Antiplatelet agents, particularly P2Y12 receptor inhibitors, are critical medicines in the prevention and treatment of thrombotic diseases in the clinic. However, their long-term use introduces a significant risk of bleeding in patients with cardiovascular diseases. Whether the bleeding is caused by the drug itself or due to surgical procedures or trauma, the need to rapidly reverse the effects of antiplatelet agents in the circulation is essential; however, no such agents are currently available. To address this need, here we describe a strategy that uses cell-membrane-wrapped nanoparticles (CM-NPs) for the rapid reversal of P2Y12 inhibitors. CM-NPs are fabricated with membranes derived from 293T cells genetically engineered to overexpress the P2Y12 receptor. Our findings support the potential of CM-NPs as a strategy for managing bleeding complications associated with P2Y12 receptor inhibitors, offering an approach to improve the safety in the use of these drugs in clinical settings.
Asunto(s)
Membrana Celular , Clopidogrel , Nanopartículas , Inhibidores de Agregación Plaquetaria , Antagonistas del Receptor Purinérgico P2Y , Receptores Purinérgicos P2Y12 , Ticagrelor , Humanos , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Ticagrelor/farmacología , Ticagrelor/química , Ticagrelor/uso terapéutico , Nanopartículas/química , Clopidogrel/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/química , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/uso terapéutico , Células HEK293RESUMEN
BACKGROUND: Peripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown. METHODS: The presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals. RESULTS: Initially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia. CONCLUSIONS: Peripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.
Asunto(s)
Cuerpo Carotídeo , Humanos , Ratones , Animales , Cuerpo Carotídeo/metabolismo , Oxígeno , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Calcio/metabolismo , Hipoxia/metabolismoRESUMEN
P2Y12 is a G-protein-coupled receptor that is activated upon ADP binding. Considering its well-established role in platelet activation, blocking P2Y12 has been used as a therapeutic strategy for antiplatelet aggregation in cardiovascular disease patients. However, receptor studies have shown that P2Y12 is functionally expressed not only in platelets and the microglia but also in other cells of the immune system, such as in monocytes, dendritic cells, and T lymphocytes. As a result, studies were carried out investigating whether therapies targeting P2Y12 could also ameliorate inflammatory conditions, such as sepsis, rheumatoid arthritis, neuroinflammation, cancer, COVID-19, atherosclerosis, and diabetes-associated inflammation in animal models and human subjects. This review reports what is known about the expression of P2Y12 in the cells of the immune system and the effect of P2Y12 activation and/or inhibition in inflammatory conditions. Lastly, we will discuss the major problems and challenges in studying this receptor and provide insights on how they can be overcome.
Asunto(s)
COVID-19 , Receptores Purinérgicos P2 , Animales , Humanos , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , COVID-19/metabolismo , Plaquetas/metabolismo , Transducción de Señal , Sistema Inmunológico , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Adenosina Difosfato/metabolismoRESUMEN
Phantom tooth pain (PTP) is a rare and specific neuropathic pain that occurs after pulpectomy and tooth extraction, but its cause is not understood. We hypothesized that there is a genetic contribution to PTP. We focused on solute carrier family 17 member 9 (SLC17A9)/vesicular nucleotide transporter (VNUT) and purinergic receptor P2Y12 (P2RY12), both of which have been associated with neuropathic pain and pain transduction signaling in the trigeminal ganglion in rodents. We sought to corroborate these associations in humans. We investigated gene polymorphisms that contribute to PTP. We statistically examined the association between genetic polymorphisms and PTP vulnerability in 150 patients with orofacial pain, including PTP, and 500 healthy subjects. We found that the rs735055 polymorphism of the SLC17A9 gene and rs3732759 polymorphism of the P2RY12 gene were associated with the development of PTP. Carriers of the minor allele of rs735055 and individuals who were homozygous for the major allele of rs3732759 had a higher rate of PTP. Carriers of the minor allele of rs735055 reportedly had high SLC17A9 mRNA expression in the spinal cord, which may increase the storage and release of adenosine triphosphate. Individuals who were homozygous for the major allele of rs3732759 may have higher P2RY12 expression that is more active in microglia. Therefore, these carriers may be more susceptible to PTP. These results suggest that specific genetic polymorphisms of the SLC17A9 and P2RY12 genes are involved in PTP. This is the first report on genes that are associated with PTP in humans.
Asunto(s)
Neuralgia , Proteínas de Transporte de Nucleótidos , Humanos , Adenosina Trifosfato/metabolismo , Proteínas de Transporte de Nucleótidos/genética , Proteínas de Transporte de Nucleótidos/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
Curcumin is a natural polyphenol derived from the turmeric plant (Curcuma longa) which exhibits numerous beneficial effects on different cell types. Inhibition of platelet activation by curcumin is well known, however molecular mechanisms of its action on platelets are not fully defined. In this study, we used laser diffraction method for analysis of platelet aggregation and Western blot for analysis of intracellular signaling mechanisms of curcumin effects on platelets. We identified two new molecular mechanisms involved in the inhibitory effects of curcumin on platelet activation. Firstly, curcumin by activation of adenosine A2A receptor stimulated protein kinase A activation and phosphorylation of Vasodilator-stimulated phosphoprotein. Secondly, we demonstrated that curcumin even at low doses, which did not inhibit platelet aggregation, potentiated inhibitory effect of ADP receptor P2Y12 antagonist cangrelor which partly could be explained by activation of adenosine A2A receptor.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Plaquetas/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Curcumina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Microfilamentos/genética , Fosfoproteínas/genética , Activación Plaquetaria/efectos de los fármacos , Receptor de Adenosina A2A/genética , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Plaquetas/citología , Plaquetas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Curcuma/química , Curcumina/aislamiento & purificación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Regulación de la Expresión Génica , Humanos , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Extractos Vegetales/química , Inhibidores de Agregación Plaquetaria/farmacología , Cultivo Primario de Células , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptor de Adenosina A2A/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Understanding how different parts of the immune system contribute to pathogenesis in Parkinson's disease is a burning challenge with important therapeutic implications. We studied enrichment of common variant heritability for Parkinson's disease stratified by immune and brain cell types. METHODS: We used summary statistics from the most recent meta-analysis of genomewide association studies in Parkinson's disease and partitioned heritability using linkage disequilibrium score regression, stratified for specific cell types, as defined by open chromatin regions. We also validated enrichment results using a polygenic risk score approach and intersected disease-associated variants with epigenetic data and expression quantitative loci to nominate and explore a putative microglial locus. RESULTS: We found significant enrichment of Parkinson's disease risk heritability in open chromatin regions of microglia and monocytes. Genomic annotations overlapped substantially between these 2 cell types, and only the enrichment signal for microglia remained significant in a joint model. We present evidence suggesting P2RY12, a key microglial gene and target for the antithrombotic agent clopidogrel, as the likely driver of a significant Parkinson's disease association signal on chromosome 3. INTERPRETATION: Our results provide further support for the importance of immune mechanisms in Parkinson's disease pathogenesis, highlight microglial dysregulation as a contributing etiological factor, and nominate a targetable microglial gene candidate as a pathogenic player. Immune processes can be modulated by therapy, with potentially important clinical implications for future treatment in Parkinson's disease. ANN NEUROL 2021;89:942-951.
Asunto(s)
Microglía/inmunología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/inmunología , Cromatina/genética , Cromosomas Humanos Par 3/genética , Clopidogrel/farmacología , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Inmunidad Celular , Desequilibrio de Ligamiento , Microglía/patología , Monocitos/patología , Herencia Multifactorial , Enfermedad de Parkinson/patología , Polimorfismo de Nucleótido Simple/genética , Receptores Purinérgicos P2Y12/genética , Medición de RiesgoRESUMEN
Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12-receptor (P2Y12R), which is expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that MPTP-induced PD symptoms in mice are associated with marked neuroinflammatory changes and P2Y12R contribute to the activation of microglia and progression of the disease. Surprisingly, while pharmacological or genetic targeting of the P2Y12R augments acute mortality in MPTP-treated mice, these interventions protect against the neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulates ROCK and p38 MAPK activity and control cytokine production. Our principal finding is that the receptor has a dualistic role in PD: functional P2Y12Rs are essential to initiate a protective inflammatory response, since the lack of the receptor leads to reduced survival; however, at later stages of neurodegeneration, P2Y12Rs are apparently responsible for maintaining the activated state of microglia and stimulating pro-inflammatory cytokine response. Understanding protective and detrimental P2Y12R-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD.
Asunto(s)
Trastornos Parkinsonianos/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/genética , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUNDS: Remarkable interindividual variability in clopidogrel response is observed, genetic polymorphisms in P2RY12 and its signal pathway is supposed to affect clopidogrel response in CHD patients. METHODS: 539 CHD patients treated with clopidogrel were recruited. The platelet reaction index (PRI) indicated by VASP-P level were detected in 12-24 h after clopidogrel loading dose or within 5-7 days after initiation of maintain dose clopidogrel. A total of 13 SNPs in relevant genes were genotyped in sample A (239 CHD patients). The SNPs which have significant differences in PRI will be validated in another sample (sample B, 300 CHD patients). RESULTS: CYP2C19*2 increased the risk of clopidogrel resistance significantly. When CYP2C19*2 and CYP2C19*3 were considered, CYP2C19 loss of function (LOF) alleles were associated with more obviously increased the risk of clopidogrel resistance; P2RY12 rs6809699C > A polymorphism was also associated with increased risk of clopidogrel resistance (AA vs CC: P = 0.0398). This difference still existed after stratification by CYP2C19 genotypes. It was also validated in sample B. The association was also still significant even in the case of stratification by CYP2C19 genotypes in all patients (sample A + B). CONCLUSION: Our data suggest that P2RY12 rs6809699 is associated with clopidogrel resistance in CHD patients. Meanwhile, the rs6809699 AA genotype can increase on-treatment platelet activity independent of CYP2C19 LOF polymorphisms.
Asunto(s)
Clopidogrel , Enfermedad Coronaria , Inhibidores de Agregación Plaquetaria , Receptores Purinérgicos P2Y12 , Humanos , Clopidogrel/farmacología , Enfermedad Coronaria/tratamiento farmacológico , Enfermedad Coronaria/genética , Citocromo P-450 CYP2C19/genética , Genotipo , Inhibidores de Agregación Plaquetaria/farmacología , Polimorfismo de Nucleótido Simple , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/genéticaRESUMEN
Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.
Asunto(s)
Plaquetas/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Adenosina Difosfato/metabolismo , Animales , Ácido Aspártico/metabolismo , Hemorragia/genética , Hemorragia/metabolismo , Humanos , Ratones , Agregación Plaquetaria , Pruebas de Función Plaquetaria , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genéticaRESUMEN
OBJECTIVE: The aim: To establish the role of allelic polymorphisms NOS3-T-786C, MTHFR-C667T, P2RY12--744C, (GPIbα)-C482T in the development of vascular lesions in patients with hypertension and diabetes mellitus type 2. PATIENTS AND METHODS: Materials and methods: The study included 100 patients with hypertension and diabetes mellitus type 2 (main group) and 50 patients without type 2 diabetes (control group). Patients underwent echocardiography, color duplex scanning of extracranial, brachiocephalic and femoral vessels. The distribution of allelic polymorphisms was investigated by isolation DNA from leukocytes and polymerase chain reaction (PCR). RESULTS: Results: The risk of vascular damages increases 2-fold when carrying all 4 risk alleles in monozygotic genotypes of polymorphic loci in patients with hypertension with concomitant type 2 diabetes (p<0,05). In gene-gene interaction, the values of contributions and directions of interaction between alleles of polymorphic loci are established (p<0,05). Genes create a paired hierarchy of interaction according to their functional activity; the largest contribution to the probable vascular damage depends on the allelic polymorphism NOS3-786CT (p<0,05), the lowest - on the allelic polymorphism P2RY12-744CC (H2H2). The genetic polymorphism of the MTHFR gene is independent of the influence of other studied polymorphisms (p<0,05); the genes P2RY12-744CT and GPIbα 482CT act synergistically with the gene NOS3-786CT, being in a weak negative interaction with each other. CONCLUSION: Conclusions: Phenotypic manifestations of endothelial dysfunction may be modified by allelic polymorphism of genes associated with endothelial and platelet functions with the risk of vascular complications.
Asunto(s)
Diabetes Mellitus Tipo 2 , Angiopatías Diabéticas , Hipertensión , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Angiopatías Diabéticas/genética , Humanos , Hipertensión/complicaciones , Hipertensión/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Óxido Nítrico Sintasa de Tipo III/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Polimorfismo Genético , Receptores Purinérgicos P2Y12/genética , Factores de RiesgoRESUMEN
Differently from other myeloid cells, microglia derive exclusively from precursors originating within the yolk sac and migrate to the CNS under development, without any contribution from fetal liver or postnatal hematopoiesis. Consistent with their unique ontology, microglia may express specific physiological markers, which have been partly described in recent years. Here we wondered whether profiles distinguishing microglia from peripheral macrophages vary with age and under pathology. To this goal, we profiled transcriptomes of microglia throughout the lifespan and included a parallel comparison with peripheral macrophages under physiological and neuroinflammatory settings using age- and sex-matched wild-type and bone marrow chimera mouse models. This comprehensive approach demonstrated that the phenotypic differentiation between microglia and peripheral macrophages is age-dependent and that peripheral macrophages do express some of the most commonly described microglia-specific markers early during development, such as Fcrls, P2ry12, Tmem119, and Trem2. Further, during chronic neuroinflammation CNS-infiltrating macrophages and not peripheral myeloid cells acquire microglial markers, indicating that the CNS niche may instruct peripheral myeloid cells to gain the phenotype and, presumably, the function of the microglia cell. In conclusion, our data provide further evidence about the plasticity of the myeloid cell and suggest caution in the strict definition and application of microglia-specific markers.SIGNIFICANCE STATEMENT Understanding the respective role of microglia and infiltrating monocytes in neuroinflammatory conditions has recently seemed possible by the identification of a specific microglia signature. Here instead we provide evidence that peripheral macrophages may express some of the most commonly described microglia markers at some developmental stages or pathological conditions, in particular during chronic neuroinflammation. Further, our data support the hypothesis about phenotypic plasticity and convergence among distinct myeloid cells so that they may act as a functional unit rather than as different entities, boosting their mutual functions in different phases of disease. This holds relevant implications in the view of the growing use of myeloid cell therapies to treat brain disease in humans.
Asunto(s)
Encéfalo/metabolismo , Diferenciación Celular/fisiología , Macrófagos/metabolismo , Microglía/metabolismo , Transcriptoma , Animales , Encéfalo/citología , Plasticidad de la Célula/fisiología , Inflamación/metabolismo , Macrófagos/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microglía/citología , Fenotipo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.
Asunto(s)
Biomarcadores/análisis , Expresión Génica , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/análisis , Placa Aterosclerótica/metabolismo , Receptores Purinérgicos P2Y12/genética , Actinas/análisis , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apolipoproteínas E/fisiología , Clopidogrel/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dieta Alta en Grasa , Técnicas de Silenciamiento del Gen , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/química , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/efectos de los fármacos , Receptores Purinérgicos P2Y12/fisiología , Transducción de Señal , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
In the past two decades, purinergic signaling has emerged as a key regulator of hematopoiesis in physiological and pathological conditions. ADP receptor P2y12 is a crucial component of this signaling, but whether it is involved in primitive hematopoiesis remains unknown. To elucidate the function of P2y12 and provide new insights for drug development, we established a zebrafish P2y12 mutant by CRISPR/Cas 9-based genetic modification system, and investigated whether P2y12 acted as an important regulator for primitive hematopoiesis. By using mass spectrometry (MS) combined with RNA sequencing, we showed that absence of P2y12 induced excessive erythropoiesis, evidenced by significantly increased expression of mature erythrocytes marker α-globin (Hbae1 and Hbae3), ß-globin (Hbbe1 and Hbbe3). Expression pattern analysis showed that P2y12 was mainly expressed in red blood cells and endothelial cells of early zebrafish embryos. Further studies revealed that primitive erythroid progenitor marker Gata1 was markedly up-regulated. Remarkably, inhibition of Gata1 by injection of Gata1 morpholino could rescue the erythroid abnormality in P2y12 mutants. The present study demonstrates the essential role of purinergic signaling in differentiation of proerythrocytes during primitive hematopoiesis, and provides potential targets for treatment of blood-related disease and drug development.
Asunto(s)
Factor de Transcripción GATA1/antagonistas & inhibidores , Hematopoyesis/fisiología , Receptores Purinérgicos P2Y12/fisiología , Proteínas de Pez Cebra/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Diferenciación Celular/fisiología , Embrión no Mamífero/fisiología , Endotelio Vascular/fisiología , Eritrocitos/fisiología , Femenino , Factor de Transcripción GATA1/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Hematopoyesis/genética , Masculino , Mutación , Receptores Purinérgicos P2Y12/genética , Regulación hacia Arriba/fisiología , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Retinal detachment (RD) is a sight-threatening complication common in many highly prevalent retinal disorders. RD rapidly leads to photoreceptor cell death beginning within 12 h following detachment. In patients with sustained RD, progressive visual decline due to photoreceptor cell death is common, leading to significant and permanent loss of vision. Microglia are the resident immune cells of the central nervous system, including the retina, and function in the homeostatic maintenance of the neuro-retinal microenvironment. It is known that microglia become activated and change their morphology in retinal diseases. However, the function of activated microglia in RD is incompletely understood, in part because of the lack of microglia-specific markers. Here, using the newly identified microglia marker P2ry12 and microglial depletion strategies, we demonstrate that retinal microglia are rapidly activated in response to RD and migrate into the injured area within 24 h post-RD, where they closely associate with infiltrating macrophages, a population distinct from microglia. Once in the injured photoreceptor layer, activated microglia can be observed to contain autofluorescence within their cell bodies, suggesting they function to phagocytose injured or dying photoreceptors. Depletion of retinal microglia results in increased disease severity and inhibition of macrophage infiltration, suggesting that microglia are involved in regulating neuroinflammation in the retina. Our work identifies that microglia mediate photoreceptor survival in RD and suggests that this effect may be due to microglial regulation of immune cells and photoreceptor phagocytosis.
Asunto(s)
Macrófagos/inmunología , Microglía/inmunología , Células Fotorreceptoras de Vertebrados/inmunología , Receptores Purinérgicos P2Y12/inmunología , Desprendimiento de Retina/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Macrófagos/patología , Ratones , Ratones Transgénicos , Microglía/patología , Células Fotorreceptoras de Vertebrados/patología , Receptores Purinérgicos P2Y12/genética , Desprendimiento de Retina/genética , Desprendimiento de Retina/patologíaRESUMEN
Epidemiological studies confirm a high risk of ischemic events in secondary-progressive multiple sclerosis (SP MS) patients, directly associated with an increased level of pro-thrombotic activity of platelets. Our work aimed to verify potential molecular abnormalities of the platelet P2Y12 receptor expression and functionality as a cause of an increased risk of thromboembolism observed in the course of MS. We have demonstrated an enhanced platelet reactivity in response to adenosine diphosphate (ADP) in SP MS relative to controls. We have also shown an increased mRNA expression for the P2RY12 gene in both platelets and megakaryocytes, as well as enhanced density of these receptors on the platelet surface. We postulate that one of the reasons for the elevated risk of ischemic events observed in MS may be a genetically or phenotypically reinforced expression of the platelet P2Y12 receptor. In order to analyze the effect of the PAR1 (protease activated receptor type 1) signaling pathway on the expression level of P2Y12, we also analyzed the correlation parameters between P2Y12 expression and the markers of platelet activation in MS induced by selective PAR1 agonist (thrombin receptor activating peptide-6, TRAP-6). Identifying the molecular base responsible for the enlarged pro-thrombotic activity of platelets in SP MS could contribute to the implementation of prevention and targeted treatment, reducing the development of cardiovascular disorders in the course of the disease.
Asunto(s)
Adenosina Difosfato/metabolismo , Esclerosis Múltiple/sangre , Activación Plaquetaria , Receptores Purinérgicos P2Y12/metabolismo , Células Cultivadas , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Receptores Purinérgicos P2Y12/genética , Transducción de SeñalRESUMEN
BACKGROUND: Microglia colonize the developing vertebrate central nervous system coincident with the detection of developmental apoptosis. Our understanding of apoptosis in intact tissue in relation to microglial clearance of dying cells is largely based on fixed samples, which is limiting given that microglia are highly motile and mobile phagocytes. Here, we used a system of microglial depletion and in vivo real-time imaging in zebrafish to directly address microglial phagocytosis of apoptotic cells during normal retinal development, the relative timing of phagocytosis in relation to apoptotic progression, and the contribution of P2RY12 signaling to this process. RESULTS: The depletion of microglia resulted in accumulation of numerous apoptotic cells in the retina. Real-time imaging revealed precise timing of microglial engulfment with the progression of apoptosis, and dynamic movement and displacement of engulfed apoptotic cells. Inhibition of P2RY12 signaling delayed microglial clearance of apoptotic cells. CONCLUSIONS: Microglial engulfment of dying cells is coincident with apoptotic progression and requires P2RY12 signaling, indicating that microglial P2RY12 signaling is shared between development and injury response. Our work provides important in vivo insight into the dynamics of apoptotic cell clearance in the developing vertebrate retina and provides a basis to understand microglial phagocytic behavior in health and disease.
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Microglía/citología , Microglía/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Retina/citología , Retina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Metronidazol/farmacología , Microglía/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Receptores Purinérgicos P2Y12/genética , Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Seizures are common in humans with various etiologies ranging from congenital aberrations to acute injuries that alter the normal balance of brain excitation and inhibition. A notable consequence of seizures is the induction of aberrant neurogenesis and increased immature neuronal projections. However, regulatory mechanisms governing these features during epilepsy development are not fully understood. Recent studies show that microglia, the brain's resident immune cell, contribute to normal neurogenesis and regulate seizure phenotypes. However, the role of microglia in aberrant neurogenic seizure contexts has not been adequately investigated. To address this question, we coupled the intracerebroventricular kainic acid model with current pharmacogenetic approaches to eliminate microglia in male mice. We show that microglia promote seizure-induced neurogenesis and subsequent seizure-induced immature neuronal projections above and below the pyramidal neurons between the DG and the CA3 regions. Furthermore, we identify microglial P2Y12 receptors (P2Y12R) as a participant in this neurogenic process. Together, our results implicate microglial P2Y12R signaling in epileptogenesis and provide further evidence for targeting microglia in general and microglial P2Y12R in specific to ameliorate proepileptogenic processes.SIGNIFICANCE STATEMENT Epileptogenesis is a process by which the brain develops epilepsy. Several processes have been identified that confer the brain with such epileptic characteristics, including aberrant neurogenesis and increased immature neuronal projections. Understanding the mechanisms that promote such changes is critical in developing therapies to adequately restrain epileptogenesis. We investigated the role of purinergic P2Y12 receptors selectively expressed by microglia, the resident brain immune cells. We report, for the first time, that microglia in general and microglial P2Y12 receptors in specific promote both aberrant neurogenesis and increased immature neuronal projections. These results indicate that microglia enhance epileptogenesis by promoting these processes and suggest that targeting this immune axis could be a novel therapeutic strategy in the clinic.
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Microglía/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Receptores Purinérgicos P2Y12/biosíntesis , Convulsiones/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/inmunología , Neuronas/inmunología , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/inmunología , Convulsiones/genética , Convulsiones/inmunologíaRESUMEN
Ticagrelor, a P2Y12-receptor inhibitor, and a non-thienopyridine agent are used to treat diabetic patients via its effects on off-target mechanisms. However, the exact sub-cellular mechanisms by which ticagrelor exerts those effects remains to be elucidated. Accordingly, the present study aimed to examine whether ticagrelor influences directly the cardiomyocytes function under insulin resistance through affecting mitochondria-sarco(endo)plasmic reticulum (SER) cross-talk. Therefore, we analyzed the function and ultrastructure of mitochondria and SER in insulin resistance-mimicked (50-µM palmitic acid for 24-h) H9c2 cardiomyocytes in the presence or absence of ticagrelor (1-µM for 24-h). We found that ticagrelor treatment significantly prevented depolarization of mitochondrial membrane potential and increases in reactive oxygen species with a marked increase in the ATP level in insulin-resistant H9c2 cells. Ticagrelor treatment also reversed the increases in the resting level of free Ca2+ and mRNA level of P2Y12 receptors as well as preserved ER stress and apoptosis in insulin-resistant H9c2 cells. Furthermore, we determined marked repression with ticagrelor treatment in the increased number of autophagosomes and degeneration of mitochondrion, including swelling and loss of crista besides recoveries in enlargement and irregularity seen in SER in insulin-resistant H9c2 cells. Moreover, ticagrelor treatment could prevent the altered mRNA levels of Becklin-1 and type 1 equilibrative nucleoside transporter (ENT1), which are parallel to the preservation of ultrastructural ones. Our overall data demonstrated that ticagrelor can directly affect cardiomyocytes and provide marked protection against ER stress and dramatic induction of autophagosomes, and therefore, can alleviate the ER stress-induced oxidative stress increase and cell apoptosis during insulin resistance.
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Apoptosis/efectos de los fármacos , Autofagosomas/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Ticagrelor/farmacología , Animales , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Beclina-1/genética , Beclina-1/metabolismo , Calcio/metabolismo , Línea Celular , Tranportador Equilibrativo 1 de Nucleósido/genética , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Insulina/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica , Mitocondrias/metabolismo , Mitocondrias/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Ácido Palmítico/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
BACKGROUND: Early neurological deterioration (END) is common in acute ischemic stroke (IS). However, the underlying mechanisms for END are unclear. The aim of this study was to evaluate the associations of 16 variants in clopidogrel-relevant genes and interactions among these variants with END in acute IS patients receiving clopidogrel treatment. METHODS: We consecutively enrolled 375 acute IS patients between June 2014 and January 2015. Platelet aggregation was measured on admission and after the 7-10 days of clopidogrel treatment. The 16 variants in clopidogrel-relevant genes were examined using mass spectrometry. The primary outcome was END within the 10 days of admission. Gene-gene interactions were analyzed by generalized multifactor dimensionality reduction (GMDR) methods. RESULTS: Among the 375 patients, 95 (25.3%) patients developed END within the first 10 days of admission. Among the 16 variants, only CYP2C19*2 (rs4244285) AA/AG was associated with END using single-locus analytical approach. GMDR analysis revealed that there was a synergistic effect of gene-gene interactions among CYP2C19*2 rs4244285, P2Y12 rs16863323, and GPIIIa rs2317676 on the risk for END. The high-risk interactions among the three variants were associated with the higher platelet aggregation and independent predictor for END after adjusting for the covariates (hazard ratio: 2.82; 95% confidence interval: 1.36-7.76; P = 0.003). CONCLUSIONS: END is very common in patients with acute IS. The mechanisms leading to END are most likely multifactorial. Interactions among CYP2C19*2 rs4244285, P2Y12 rs16863323, and GPIIIa rs2317676 may confer a higher risk for END. It was very important to modify clopidogrel therapy for the patients carrying the high-risk interactive genotypes. CLINICAL TRIAL REGISTRATION INFORMATION: The study described here is registered at http://www.chictr.org/ (unique Identifier: ChiCTR-OCH-14004724). The date of trial registration was May 30, 2014.
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Isquemia Encefálica/genética , Clopidogrel/efectos adversos , Citocromo P-450 CYP2C19/genética , Epistasis Genética/genética , Integrina beta3/genética , Agregación Plaquetaria/genética , Receptores Purinérgicos P2Y12/genética , Recurrencia , Accidente Cerebrovascular/genética , Isquemia Encefálica/complicaciones , Isquemia Encefálica/tratamiento farmacológico , Clopidogrel/uso terapéutico , Genotipo , Humanos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/efectos adversos , Polimorfismo de Nucleótido Simple/genética , Estudios Prospectivos , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológicoRESUMEN
PURPOSE: Dual antiplatelet therapy (DAT) with clopidogrel and aspirin is not suitable for clopidogrel resistance (CR) patients with aspirin intolerance. To investigate the prevalence of CR in patients with aspirin intolerance after ischemic stroke (IS) and to assess the relationship between CR and CYP2C19, P2Y12 receptor genotypes in patients with aspirin intolerance after IS. METHODS: We enrolled 126 IS patients with aspirin intolerance from Han Chinese in Shangqiu from January 2016 to November 2018. All IS patients with aspirin intolerance were treated with clopidogrel for 7 days. Adenosine diphosphate-induced platelet inhibition rate was measured by thrombelastography (TEG) mapping assay. The SNPs CYP2C19*2, CYP2C19*3, and P2Y12 receptor (52 G >T) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Binary logistic regression analyses were performed using SPSS version 20.0. RESULTS: The prevalence of CR in patients with aspirin intolerance after IS was approximately 31.0%. Multivariate regression analysis showed that body weight (OR 1.091 (95% CI 1.031-1.155), p = 0.003), CYP2C19 phenotype intermediate metabolizer (IM) (OR 3.820 (95% CI 1.021-14.288), p = 0.046), and CYP2C19 phenotype poor metabolizer (PM) (OR 14.481 (95% CI 2.791-75.129), p = 0.001) significantly increased the risk of CR and P2Y12 receptors (52 G >T) (OR 3.498 [95% CI 1.251-9.784], p = 0.017) increased the risk of CR. CONCLUSIONS: The patients with high body weight, the CYP2C19 phenotypes, and P2Y12 receptor (52 G >T) variant alleles are at risk of CR during clopidogrel treatment in Chinese IS patients with aspirin intolerance. The higher body weight and relevant polymorphisms may help to predict CR in Chinese IS patients with aspirin intolerance.