RESUMO
Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.
Assuntos
Bevacizumab/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/efeitos adversos , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/uso terapêutico , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Bevacizumab/efeitos adversos , Bevacizumab/uso terapêutico , Linhagem Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Degeneração Macular/terapia , Camundongos , Camundongos Transgênicos , Ativação Plaquetária , Ligação Proteica , Multimerização Proteica , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/etiologia , Trombose/etiologia , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
The p75 neurotrophin receptor is important in multiple physiological actions including neuronal survival and neurite outgrowth during development, and after central nervous system injury. We have discovered a novel piperazine-derived compound, EVT901, which interferes with p75 neurotrophin receptor oligomerization through direct interaction with the first cysteine-rich domain of the extracellular region. Using ligand binding assays with cysteine-rich domains-fused p75 neurotrophin receptor, we confirmed that EVT901 interferes with oligomerization of full-length p75 neurotrophin receptor in a dose-dependent manner. Here we report that EVT901 reduces binding of pro-nerve growth factor to p75 neurotrophin receptor, blocks pro-nerve growth factor induced apoptosis in cells expressing p75 neurotrophin receptor, and enhances neurite outgrowth in vitro Furthermore, we demonstrate that EVT901 abrogates p75 neurotrophin receptor signalling by other ligands, such as prion peptide and amyloid-ß. To test the efficacy of EVT901 in vivo, we evaluated the outcome in two models of traumatic brain injury. We generated controlled cortical impacts in adult rats. Using unbiased stereological analysis, we found that EVT901 delivered intravenously daily for 1 week after injury, reduced lesion size, protected cortical neurons and oligodendrocytes, and had a positive effect on neurological function. After lateral fluid percussion injury in adult rats, oral treatment with EVT901 reduced neuronal death in the hippocampus and thalamus, reduced long-term cognitive deficits, and reduced the occurrence of post-traumatic seizure activity. Together, these studies provide a new reagent for altering p75 neurotrophin receptor actions after injury and suggest that EVT901 may be useful in treatment of central nervous system trauma and other neurological disorders where p75 neurotrophin receptor signalling is affected.
Assuntos
Oligodendroglia/efeitos dos fármacos , Piperazinas/farmacologia , Receptor de Fator de Crescimento Neural/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doenças Desmielinizantes/patologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oligodendroglia/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Ensaio Radioligante , Ratos , Receptor de Fator de Crescimento Neural/biossíntese , Receptor trkA/metabolismo , Recuperação de Função FisiológicaRESUMO
RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/- and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18.
Assuntos
Megacariócitos/metabolismo , Ativação Plaquetária/fisiologia , Proteínas RGS/genética , Proteínas RGS/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Contagem de Células Sanguíneas , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Filogenia , Ativação Plaquetária/genética , Regiões Promotoras Genéticas/genética , Serotonina/sangue , Transdução de Sinais/genética , Trombose/metabolismoRESUMO
In the search of a potential backup for clopidogrel, we have initiated a HTS campaign designed to identify novel reversible P2Y12 antagonists. Starting from a hit with low micromolar binding activity, we report here the main steps of the optimization process leading to the identification of the preclinical candidate SAR216471. It is a potent, highly selective, and reversible P2Y12 receptor antagonist and by far the most potent inhibitor of ADP-induced platelet aggregation among the P2Y12 antagonists described in the literature. SAR216471 displays potent in vivo antiplatelet and antithrombotic activities and has the potential to differentiate from other antiplatelet agents.
Assuntos
Indóis/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2/farmacologia , Piridazinas/farmacologia , Receptores Purinérgicos P2Y12/metabolismo , Síndrome Coronariana Aguda/prevenção & controle , Difosfato de Adenosina/farmacologia , Administração Oral , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Humanos , Indóis/síntese química , Indóis/metabolismo , Injeções Intravenosas , Masculino , Modelos Químicos , Estrutura Molecular , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/metabolismo , Antagonistas do Receptor Purinérgico P2/síntese química , Antagonistas do Receptor Purinérgico P2/metabolismo , Piridazinas/síntese química , Piridazinas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y12/genética , Trombose/prevenção & controleRESUMO
Myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) represent prominent components in cancer progression. We previously showed that inhibition of the VEGFR-3 pathway by SAR131675 leads to reduction of TAM infiltration and tumor growth. Here, we found that treatment with SAR131675 prevents the accumulation of immunosuppressive blood and splenic MDSCs which express VEGFR-3, in 4T1 tumor bearing mice. Moreover we showed that soluble factors secreted by tumor cells promote MDSCs proliferation and differentiation into M2 polarized F4/80+ macrophages. In addition, cell sorting and transcriptomic analysis of tumor infiltrating myeloid cells revealed the presence of a heterogeneous population that could be divided into 3 subpopulations: (i) immature cells with a MDSC phenotype (GR1+/CD11b+/F4/80-); (ii) "immuno-incompetent" macrophages (F4/80high/CD86neg/MHCIILow) strongly expressing M2 markers such as Legumain, CD206 and Mgl1/2 and (iii) "immuno-competent"-M1 like macrophages (F4/80Low/CD86+/MHCIIHigh). SAR131675 treatment reduced MDSCs in lymphoid organs as well as F4/80High populations in tumors. Interestingly, in the tumor SAR131675 was able to increase the immunocompetent M1 like population (F4/80low). Altogether these results demonstrate that the specific VEGFR-3 inhibitor SAR131675 exerts its anti tumoral activity by acting on different players that orchestrate immunosuppression and cancer progression in a tumoral context: MDSCs in peripheral lymphoid organs and TAMs infiltrating the tumor.
RESUMO
SAR131675 is a potent and selective VEGFR-3 inhibitor. It inhibited VEGFR-3 tyrosine kinase activity and VEGFR-3 autophosphorylation in HEK cells with IC(50) values of 20 and 45 nmol/L, respectively. SAR131675 dose dependently inhibited the proliferation of primary human lymphatic cells, induced by the VEGFR-3 ligands VEGFC and VEGFD, with an IC(50) of about 20 nmol/L. SAR131675 was found to be highly selective for VEGFR-3 versus 107 receptors, enzymes, ion channels, and 65 kinases. However, it was moderately active on VEGFR-2 with a VEGFR-3/VEGFR-2 ratio of about 10. SAR131675 had no antiproliferative activity on a panel of 30 tumors and primary cells, further showing its high specificity and indicating that SAR131675 is not a cytotoxic or cytostatic agent. SAR131675 was very well tolerated in mice and showed a potent antitumoral effect in several orthotopic and syngenic models, including mammary 4T1 carcinoma and RIP1.Tag2 tumors. Interestingly, it significantly reduced lymph node invasion and lung metastasis, showing its antilymphangiogenic activity in vivo. Moreover, treatment of mice before resection of 4T1 primary tumors was sufficient to prevent metastasis. Tumor-associated macrophages (TAM) play an important role in tumor growth and metastasis. The expression of VEGFR-3 on TAMs has been recently described. F4/80 immunostaining clearly showed that SAR131675 significantly reduced TAM infiltration and aggregation in 4T1 tumors. Taken together, SAR131675 is the first highly specific VEGFR-3-TK inhibitor described to date, displaying significant antitumoral and antimetastatic activities in vivo through inhibition of lymphangiogenesis and TAM invasion.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Linfangiogênese/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Macrófagos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Naftiridinas/administração & dosagem , Metástase Neoplásica , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Communication between endothelial cells and cardiomyocytes is critical for cardiac development and regeneration. However the mechanisms involved in these endothelial-cardiomyocyte interactions remain poorly understood. Nucleotides are released within the heart, especially under ischemia or pressure overload. The function of P2Y nucleotide receptors in cardiac development has never been investigated. Here we show that adult P2Y(4)-null mice display microcardia. P2Y(4) nucleotide receptor is expressed in cardiac endothelial cells but not in cardiomyocytes. Loss of P2Y(4) in cardiac endothelial cells strongly inhibits their growth, migration and PDGF-B secretion in response to UTP. Proliferation of microvessels and cardiomyocytes is reduced in P2Y(4)-null hearts early after birth, resulting in reduced heart growth. Our study uncovers mouse P2Y(4) receptor as an essential regulator of cardiac endothelial cell function, and illustrates the involvement of endothelial-cardiomyocyte interactions in post-natal heart development. We also detected P2Y(4) expression in human cardiac microvessels. P2Y(4) receptor could constitute a therapeutic target to regulate cardiac remodelling and post-ischemic revascularisation.
Assuntos
Coração/crescimento & desenvolvimento , Receptores Purinérgicos P2/fisiologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Endothelial cells (ECs) are the primary sensors of variations in blood oxygen concentrations. They use the hypoxia-sensitive stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription factor to engage specific transcriptional programs in response to oxygen changes. The regulation of HIF-1α expression is well documented at the protein level, but much less is known about the control of its mRNA stability. Using small interfering RNA knockdown experiments, reporter gene analyses, ribonucleoprotein immunoprecipitations, and mRNA half-life determinations, we report a new regulatory mechanism of HIF-1α expression in ECs. We demonstrate that 1) sustained hypoxia progressively decreases HIF-1α mRNA while HIF-1α protein levels rapidly peak after 3 h and then slowly decay; 2) silencing the mRNA-destabilizing protein tristetraprolin (TTP) in ECs reverses hypoxia-induced down-regulation of HIF-1α mRNA; 3) the decrease in the half-life of Luciferase-HIF-1α-3'UTR reporter transcript that is observed after prolonged hypoxia is mediated by TTP; 4) TTP binds specifically to HIF-1α 3'UTR; and 5) the most distal AU-rich elements present in HIF-1α 3'UTR (composed of two hexamers) are sufficient for TTP-mediated repression. Finally, we bring evidence that silencing TTP expression enhances hypoxia-induced increase in HIF-1α protein levels with a concomitant increase in the levels of the carbonic anhydrase enzyme CA IX, thus suggesting that TTP physiologically controls the expression of a panel of HIF-1α target genes. Altogether, these data reveal a new role for TTP in the control of gene expression during the response of endothelial cell to hypoxia.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Tristetraprolina/metabolismo , Regiões 3' não Traduzidas , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sequência de Bases , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Hipóxia Celular/genética , Linhagem Celular , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Genes Reporter , Meia-Vida , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Tristetraprolina/genéticaRESUMO
Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of [³³P]-2-methylthio-ADP on P2Y12-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC50: CHO-P2Y12: 0.12 µM; PRP: 0.97 µM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.
Assuntos
Plaquetas/metabolismo , Plasma/citologia , Receptores Purinérgicos P2Y12/metabolismo , Ticlopidina/análogos & derivados , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Animais , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Células CHO , Cromatografia Líquida de Alta Pressão , Clopidogrel , Cricetinae , Cricetulus , Humanos , Espectrometria de Massas , Isótopos de Fósforo/química , Plasma/química , Vigilância de Produtos Comercializados/métodos , Ligação Proteica/efeitos dos fármacos , Receptores Purinérgicos P2Y12/genética , Sensibilidade e Especificidade , Estereoisomerismo , Tionucleotídeos/química , Tionucleotídeos/metabolismo , Ticlopidina/análise , Ticlopidina/química , Ticlopidina/farmacologia , Transgenes/genéticaRESUMO
Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF.FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM(6)), octasaccharide (HM(8)), and decasaccharide (HM(10)), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM(8) and HM(10) are significantly more potent than HM(6) in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1.FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2.FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1.FGFR4 interaction site and a direct FGFR4.FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF.FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM(8) relative to HM(6) in stimulating FGF2.FGFR4 signaling correlates with the higher affinity of HM(8) to bind and dimerize FGF2. Notably FGF2.HM(8) exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF.FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Heparina/análogos & derivados , Oligossacarídeos/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Camundongos , Complexos Multiproteicos/biossíntese , Oligossacarídeos/química , Ligação Proteica , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
HLA-G is a major histocompatibility complex class Ib molecule whose constitutive tissue distribution is restricted mainly to trophoblast cells at the maternal-fetal interface during pregnancy. In this study, we demonstrated the ability of the soluble HLA-G1 (sHLA-G1) isoform to inhibit fibroblast growth factor-2 (FGF2)-induced capillary-like tubule formation. Using a rabbit corneal neovascularization model, we further showed that sHLA-G1 inhibits FGF2-induced angiogenesis in vivo. We also demonstrated that sHLA-G1 induces endothelial cell apoptosis through binding to BY55/CD160, a glycosylphosphatidylinositolanchored receptor expressed by endothelial cells. Furthermore, we showed that the specific CL1-R2 anti-CD160 monoclonal antibody mimics sHLA-G1-mediated inhibition of endothelial cell tube formation and induction of apoptosis. Thus, the engagement of CD160 in endothelial cells may be essential for the inhibition of angiogenesis. sHLA-G1/CD160-mediated antiangiogenic property may participate in the vascular remodeling of maternal spiral arteries during pregnancy, and, given that we found that CD160 is strongly expressed in the vasculature of a murine tumor, it offers an attractive therapeutic target for preventing pathologic neovascularization.
Assuntos
Antígenos CD/metabolismo , Células Endoteliais/efeitos dos fármacos , Antígenos HLA/metabolismo , Antígenos HLA/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Apoptose/fisiologia , Células Cultivadas , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/imunologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Ligadas por GPI , Antígenos HLA-G , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Coelhos , Receptores Imunológicos/genética , SolubilidadeRESUMO
P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12/P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met.
Assuntos
Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Receptores Purinérgicos P2/metabolismo , Ticlopidina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Clopidogrel , Humanos , Mutação/genética , Ligação Proteica , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y12 , Ticlopidina/metabolismo , Ticlopidina/farmacologiaRESUMO
Neuropilin 2 (NRP2) is a receptor for the vascular endothelial growth factor (VEGF) and the semaphorin (SEMA) families, 2 unrelated ligand families involved in angiogenesis and neuronal guidance. NRP2 specifically binds VEGF-A and VEGF-C, although the biological relevance of these interactions in human endothelial cells is poorly understood. In this study, we show that both VEGF-A and VEGF-C induce the interaction of NRP2 with VEGFR-2. This interaction correlated with an enhancement of the VEGFR-2 phosphorylation threshold. Overexpression of NRP2 in primary human endothelial cells promoted cell survival induced by VEGF-A and VEGF-C. In contrast, SEMA3F, another ligand for NRP2, was able to inhibit human endothelial cell survival and migration induced by VEGF-A and VEGF-C. Moreover, a siRNA targeting specifically NRP2 was a potent inhibitor of human endothelial cell migration induced by VEGF-A and VEGF-C. Thus, our data indicate that NRP2 acts as a coreceptor that enhances human endothelial cell biological responses induced by VEGF-A and VEGF-C.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Neuropilina-2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Neuropilina-2/antagonistas & inibidores , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Patients suffering a transient ischaemic attack (TIA) or ischaemic stroke (IS) have a high risk of recurrence. The inhibition of platelet function is effective in the reduction of secondary vascular events in patients with TIA or stroke. This is true for acetylsalicylic acid (ASA), clopidogrel, ticlopidine and the combination of ASA plus slow-release dipyridamole. This overview analyses the results of recent trials and presents ongoing or future trials with clopidogrel as well as the combination of clopidogrel plus ASA. Clopidogrel is superior to ASA in the prevention of vascular events in patients with IS, myocardial infarction (MI) or peripheral arterial disease (PAD). The difference is highest for high-risk patients such as diabetics, patients who underwent coronary bypass surgery and patients with a remote prior history of ischaemic events. A prediction model is presented which allows the identification of patients in whom clopidogrel is superior to ASA for the secondary prevention of stroke. The combination of clopidogrel and ASA is better than ASA alone in patients undergoing coronary stent implantations and patients with unstable angina or non-Q-wave MI. In high-risk patients with TIA or stroke, the addition of ASA to clopidogrel is not superior to ASA monotherapy but results in a higher rate of bleeding complications. The long-term combination therapy is currently investigated in several large trials in > 30,000 patients, with a large number of stroke patients.
Assuntos
Acidente Vascular Cerebral/prevenção & controle , Ticlopidina/análogos & derivados , Ensaios Clínicos como Assunto/estatística & dados numéricos , Clopidogrel , Humanos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/uso terapêutico , Fatores de Risco , Acidente Vascular Cerebral/sangue , Ticlopidina/química , Ticlopidina/uso terapêuticoRESUMO
Ticlopidine and clopidogrel belong to the same chemical family of thienopyridine adenosine diphosphate (ADP)-receptor antagonists. They have shown their efficacy as platelet antiaggregant and antithrombotic agents in many animal models, both ex vivo and in vivo. Although ticlopidine was discovered more than 30 years ago, it was only recently that the mechanism of action of ADP-receptor antagonists was characterized in detail. Ticlopidine and clopidogrel both behave in vivo as specific antagonists of P2Y (12), one of the ADP receptors on platelets. Metabolic steps that involve cytochrome P450-dependent pathways are required to generate the active metabolite responsible for this in vivo activity. The active moiety is a reactive thiol derivative that targets P2Y (12) on platelets. The interaction is irreversible, accounting for the observation that platelets are definitely antiaggregated, even if no active metabolite is detectable in plasma. The interaction is specific for P2Y (12); other purinoceptors such as P2Y (1) and P2Y (13) are spared. This results in inhibition of the binding of the P2Y (12) agonist 2-methylthio-ADP and the ADP-induced downregulation of adenylyl cyclase. Platelet aggregation is affected not only when triggered by ADP but also by aggregation inducers when used at concentrations requiring released ADP as an amplifier. The efficacy and safety of clopidogrel has been established in several large, randomized, controlled trials. The clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE) trial demonstrated the superiority of clopidogrel over acetylsalicylic acid (ASA) in patients at risk of ischemic events, including ischemic stroke, myocardial infarction (MI), and peripheral arterial disease. The clopidogrel in unstable angina to prevent recurrent ischemic events (CURE) trial showed a sustained, incremental benefit when clopidogrel was added to standard therapy (including ASA) in patients with unstable angina and non-Q-wave MI. The clopidogrel for the reduction of events during observation (CREDO) trial demonstrated the benefit of continuing clopidogrel (plus ASA) for 12 months, as opposed to 1 month, after percutaneous coronary intervention. The proven efficacy of clopidogrel, coupled with its favorable safety and tolerability profile, has prompted its evaluation in an extensive, ongoing clinical trial program that will help to further characterize the benefit of clopidogrel in patients with a range of atherothrombotic profiles.
Assuntos
Arteriosclerose/complicações , Fibrinolíticos/uso terapêutico , Proteínas de Membrana/antagonistas & inibidores , Inibidores da Agregação Plaquetária/uso terapêutico , Antagonistas do Receptor Purinérgico P2 , Trombofilia/tratamento farmacológico , Trombose/prevenção & controle , Ticlopidina/análogos & derivados , Ticlopidina/uso terapêutico , Difosfato de Adenosina/farmacologia , Animais , Aspirina/farmacologia , Aspirina/uso terapêutico , Ensaios Clínicos como Assunto , Clopidogrel , Estudos de Coortes , Método Duplo-Cego , Resistência a Medicamentos , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacologia , Previsões , Humanos , Metanálise como Assunto , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/farmacologia , Piridinas/farmacologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Ratos , Receptores Purinérgicos P2 , Receptores Purinérgicos P2Y12 , Trombofilia/etiologia , Trombose/etiologia , Ticlopidina/farmacocinética , Ticlopidina/farmacologiaRESUMO
The dual role of P2Y1 and P2Y12 receptors in platelet aggregation by ADP has been firmly established, based on the action of selective inhibitors, gene targeting in mice and human genetic evidence. Both of these receptor subtypes constitute targets for antithrombotic agents, and compounds with a dual action might also be of interest. However, the agents currently on the market (ticlopidine and clopidogrel), or known to be in development (cangrelor, AZD-6140 and prasugrel), all target the P2Y12 receptor. The thienopyridines (ticlopidine, clopidogrel and prasugrel) irreversibly inactivate the P2Y12 receptor via the covalent binding of an active metabolite generated in the liver, while the other compounds are competitive antagonists. Cangrelor, an ATP derivative, is suitable for intravenous perfusion, whereas AZD-6140 is in clinical development as an orally active agent.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Trombose/tratamento farmacológico , Ticlopidina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Animais , Clopidogrel , Humanos , Camundongos , Ticlopidina/química , Ticlopidina/farmacologiaRESUMO
The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Isquemia/tratamento farmacológico , Isquemia/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Humanos , Isquemia/induzido quimicamente , Isquemia/metabolismo , Linfocinas , Camundongos , Microcirculação/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Fosfotirosina/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Células-Tronco/citologiaRESUMO
Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing "outside-in" signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the beta3 integrin, thereby amplifying outside-in signaling via alphaIIbbeta3. Blocking the Gas6-R-alphaIIbbeta3 integrin cross-talk might be a novel approach to the reduction of thrombosis.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Agregação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Trombose/metabolismo , Animais , Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores Proteína Tirosina Quinases/administração & dosagem , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Trombose/tratamento farmacológico , Trombose/genética , Trombose/patologiaRESUMO
Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy caused by antibodies against a complex of platelet factor 4 and heparin. Fondaparinux (Arixtra) is a new synthetic selective factor Xa inhibitor. We performed a serologic study to determine the cross-reactivity of HIT sera with fondaparinux. Using a prospective, blinded study design, 39 clinically and serologically confirmed sera from patients with HIT and 15 control sera were sent to 3 different laboratories, each of which specialized in a particular HIT assay. These include the serotonin release assay, heparin-induced platelet agglutination assay, and platelet aggregation assay. Two of 82 assays (2.4%) performed in the presence of control sera were positive, both with unfractionated heparin. In the presence of HIT sera, 75 of 94 (79.8%) evaluable assays were positive with unfractionated heparin; fondaparinux was significantly (P < .001) less reactive than unfractionated heparin, only 3 of 91 evaluable assays (3.3%) being positive. Using flow cytometry, unlike unfractionated heparin, fondaparinux did not induce the binding of PAC1 and anti-CD62 monoclonal antibodies or of annexin V to platelets with HIT sera. Together, these results suggest that fondaparinux is nonreactive to HIT sera and raise the possibility that the drug may be used for prophylaxis and treatment of thrombosis in patients with a history of HIT.
Assuntos
Autoanticorpos/imunologia , Heparina/imunologia , Heparina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Polissacarídeos/farmacologia , Citometria de Fluxo , Fondaparinux , Humanos , Serotonina/sangue , Método Simples-Cego , Trombocitose/sangue , Trombocitose/induzido quimicamente , Trombocitose/complicações , Trombocitose/tratamento farmacológicoRESUMO
VEGFR-3 is essential for vascular development and maintenance of lymphatic vessel's integrity. Little is known about its cooperative effect with other receptors of the same family. Contrary to VEGFR-2, stimulation of VEGFR-3 by VEGF-C and -D failed to enhance its phosphorylation either in HEK293T or in PAE cells. These ligands were unable to induce angiogenesis of PAEC expressing VEGFR-3 alone. In the presence of VEGFR-2, VEGF-C and -D induced heterodimerization of VEGFR-3 with VEGFR-2. This heterodimerization was associated with enhanced VEGFR-3 phosphorylation and subsequent cellular responses as evidenced by the formation of capillary-like structures in PAE cells and proliferation of primary human endothelial cells expressing both receptors. Taken together, these results show for the first time that VEGFR-3 needs to be associated to VEGFR-2 to induce ligand-dependent cellular responses.