RESUMO
Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.
Assuntos
Fator Plaquetário 4 , Mielofibrose Primária , Proteômica , Fator Plaquetário 4/metabolismo , Fator Plaquetário 4/genética , Animais , Camundongos , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/genética , Humanos , Proteômica/métodos , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos Endogâmicos C57BL , Diferenciação CelularRESUMO
PURPOSE OF REVIEW: Myelofibrosis (MF) is primarily driven by constitutive activation of the Janus kinase/signal transducer of activators of transcription (JAK/STAT) pathway. While JAK inhibitors have shown to alleviate disease symptoms, their disease-modifying effects in MF are limited. The only curative treatment remains allogeneic stem cell transplantation, which can be applied to a minority of patients. As a result, there is a need to explore novel targets in MF to facilitate appropriate drug development and therapeutic pathways. RECENT FINDINGS: Recent research has focused on identifying novel signals that contribute to the abnormal cross-talk between hematopoietic and stromal cells, which promotes MF and disease progression. Inflammation and immune dysregulation have emerged as key drivers of both the initiation and progression of MF. A growing number of actionable targets has been identified, including cytokines, transcription factors, signalling networks and cell surface-associated molecules. These targets exhibit dysfunctions in malignant and nonmalignant hematopoietic cells, but also in nonhematopoietic cells of the bone marrow. The study of these inflammation-related molecules, in preclinical models and MF patient's samples, is providing novel therapeutic targets. SUMMARY: The identification of immunotherapeutic targets is expanding the therapeutic landscape of MF. This review provides a summary of the most recent advancements in the study of immunotherapeutic targets in MF.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Mielofibrose Primária , Humanos , Mielofibrose Primária/tratamento farmacológico , Janus Quinases , Imunoterapia , Janus Quinase 2RESUMO
The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."
Assuntos
Encefalopatias , Moléculas de Adesão Celular , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Animais , Camundongos , Alelos , Encefalopatias/genética , Moléculas de Adesão Celular/genética , Células Endoteliais/metabolismo , Hemorragias Intracranianas/genética , Malformações do Sistema Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Junções Íntimas/genética , HumanosRESUMO
Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-/- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-/- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-/- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-/- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-/-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.
Assuntos
Autofagia , Fosfatidilinositol 3-Quinases , Camundongos , Animais , Autofagia/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Megacariócitos/metabolismo , Colágeno Tipo VI , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático , Chaperona BiP do Retículo Endoplasmático , Proteínas Proto-Oncogênicas c-bcl-2 , SirolimoRESUMO
In primary myelofibrosis, extra-domain A fibronectin (EDA-FN), the result of alternative splicing of FN gene, sustains megakaryocyte proliferation and confers a pro-inflammatory phenotype to bone marrow cell niches. In this work we assessed the levels of circulating EDA-FN in plasma samples of 122 patients with primary myelofibrosis. Patients with a homozygous JAK2V617F genotype displayed the higher level of plasma EDA-FN. Increased EDA-FN levels were associated with anemia, elevated high-sensitivity C-reactive protein, bone marrow fibrosis and splanchnic vein thrombosis at diagnosis. While no correlation was observed with CD34+ hematopoietic stem cell mobilization, elevated blood level of EDA-FN at diagnosis was a predictor of large splenomegaly (over 10 cm from the left costal margin) outcome. Thus, EDA-FN expression in primary myelofibrosis may represent the first marker of disease progression, and a novel target to treat splenomegaly.
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Depression is associated with thrombotic risk and arterial events, its proper management is strongly recommended in coronary artery disease (CAD) patients. We have previously shown that the Brain-Derived Neurotrophic Factor (BDNF)Val66Met polymorphism, related to depression, is associated with arterial thrombosis in mice, and with an increased risk of acute myocardial infarction in humans. Herein, expanding the previous findings on BDNFVal66Met polymorphism, we show that desipramine, a norepinephrine reuptake-inhibitor, rescues behavioral impairments, reduces the arterial thrombosis risk, abolishes pathological coagulation and platelet hyper-reactivity, normalizes leukocyte, platelet, and bone marrow megakaryocyte number and restores physiological norepinephrine levels in homozygous knock-in BDNF Val66Met (BDNFMet/Met) mice. The in vitro data confirm the enhanced procoagulant activity and the alpha2A-adrenergic receptor (α2A-ADR) overexpression found in BDNFMet/Met mice and we provide evidence that, in presence of Met variant, norepinephrine is crucial to up-regulate procoagulant activity and to enhance platelet generation. The α2-ADR antagonist rauwolscine rescues the prothrombotic phenotype in BDNFMet/Met mice and reduces procoagulant activity and platelet generation in cells transfected with BDNFMet plasmid or exposed to pro-BDNFMet peptide. Finally, we show that homozygous BDNFMet/Met CAD patients have hyper-reactive platelets overexpressing abundant α2A-ADR. The great proplatelet release from their megakaryocytes well reflects their higher circulating platelet number compared to BDNFVal/Val patients. These data reveal an unprecedented described role of Met allele in the dysregulation of norepinephrine/α2A-ADR pathway that may explain the predisposition to arterial thrombosis. Overall, the development of α2A-ADR inhibitors might represent a pharmacological treatment for depression-associated thrombotic conditions in this specific subgroup of CAD patients.
Assuntos
Coagulação Sanguínea/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Depressão/patologia , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Trombose/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Doença da Artéria Coronariana/patologia , Desipramina/farmacologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Thrombopoiesis governs the formation of blood platelets in bone marrow by converting megakaryocytes into long, branched proplatelets on which individual platelets are assembled. The megakaryocyte cytoskeleton responds to multiple microenvironmental cues, including chemical and mechanical stimuli, sustaining the platelet shedding. During the megakaryocyte's life cycle, cytoskeletal networks organize cell shape and content, connect them physically and biochemically to the bone marrow vascular niche, and enable the release of platelets into the bloodstream. While the basic building blocks of the cytoskeleton have been studied extensively, new sets of cytoskeleton regulators have emerged as critical components of the dynamic protein network that supports platelet production. Understanding how the interaction of individual molecules of the cytoskeleton governs megakaryocyte behavior is essential to improve knowledge of platelet biogenesis and develop new therapeutic strategies for inherited thrombocytopenias caused by alterations in the cytoskeletal genes.
Assuntos
Proteínas do Citoesqueleto , Megacariócitos , Plaquetas/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Megacariócitos/metabolismo , TrombopoeseRESUMO
In this chapter, we will discuss the current knowledge concerning the alterations of the cellular components in the bone marrow niche in Myeloproliferative Neoplasms (MPNs), highlighting the central role of the megakaryocytes in MPN progression, and the extracellular matrix components characterizing the fibrotic bone marrow.
Assuntos
Medula Óssea , Transtornos Mieloproliferativos , Células da Medula Óssea , Humanos , MegacariócitosRESUMO
Spontaneous cervical artery dissection (CeAD) is a major cause of ischemic stroke in young adults, whose genetic susceptibility factors are still largely unknown. Nevertheless, subtle ultrastructural connective tissue alterations (especially in the collagen fibril morphology) are recognized in a large proportion of CeAD patients, in which recent genetic investigations reported an enrichment of variants in genes associated with known connective tissue disorders. In this regard, COL5A1 variants have been reported in a small subset of CeAD patients, with or without classical Ehlers-Danlos syndrome (cEDS) features. We investigated a 22-year-old patient with intracranial aneurysm and mild connective tissue manifestations reminiscent of EDS. Whole-exome sequencing identified two COL5A1 missense variants in trans configuration: NM_000093.5:c.[1588G>A];[4135C>T], NP_000084.3:p.[(Gly530Ser)];[(Pro1379Ser)]. Functional assays demonstrated a significant decrease of collagen α1(V) chain expression in both heterozygous parents compared to control cells, and an additive effect of these two variants in the proband. Interestingly, both parents manifested very subtle EDS signs, such as atrophic scars, recurrent bone fractures, colonic diverticulosis, varicose veins, and osteoarthritis. Our findings emphasize the involvement of COL5A1 in the predisposition to vascular phenotypes and provide novel insights on the c.1588G>A variant, whose functional significance has not been definitely established. In fact, it was previously reported as both "disease modifying", and as a biallelic causative mutation (with heterozygous individuals showing subtle clinical signs of cEDS). We speculated that the c.1588G>A variant might lead to overt phenotype in combination with additional genetic "hits" lowering the collagen α1(V) chain expression below a hypothetical disease threshold.
Assuntos
Colágeno Tipo V/genética , Síndrome de Ehlers-Danlos/genética , Aneurisma Intracraniano/genética , Penetrância , Tecido Conjuntivo/patologia , Síndrome de Ehlers-Danlos/patologia , Humanos , Aneurisma Intracraniano/patologia , Masculino , Mutação de Sentido Incorreto , Adulto JovemRESUMO
Mechanisms related to platelet release in the context of the bone marrow niche are not completely known. In this review we discuss what has been discovered about four critical aspects of this process: 1) the bone marrow niche organization, 2) the role of the extracellular matrix components, 3) the mechanisms by which megakaryocytes release platelets and 4) the novel approaches to mimic the bone marrow environment and produce platelets ex vivo.
Assuntos
Plaquetas/metabolismo , Animais , HumanosRESUMO
Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.
Assuntos
Integrina alfa5beta1/fisiologia , Megacariócitos/patologia , Mielofibrose Primária/patologia , Animais , Medula Óssea/metabolismo , Adesão Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Humanos , Integrina alfa5/biossíntese , Integrina alfa5/genética , Integrina alfa5/imunologia , Integrina alfa5beta1/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Mielofibrose Primária/genéticaRESUMO
The fibronectin EDA isoform (EDA FN) is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We found that mice constitutively expressing the EDA domain (EIIIA+/+), but not EDA knockout mice, are more prone to develop BM fibrosis upon treatment with the thrombopoietin (TPO) mimetic romiplostim (TPOhigh). Mechanistically, EDA FN binds to TLR4 and sustains progenitor cell proliferation and megakaryopoiesis in a TPO-independent fashion, inducing LPS-like responses, such as NF-κB activation and release of profibrotic IL-6. Pharmacological inhibition of TLR4 or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis, and splenomegaly. Finally, developing a novel ELISA assay, we analyzed samples from patients affected by primary myelofibrosis (PMF), a well-known pathological situation caused by altered TPO signaling, and found that the EDA FN is increased in plasma and BM biopsies of PMF patients as compared with healthy controls, correlating with fibrotic phase.
Assuntos
Fibronectinas/sangue , Fibronectinas/metabolismo , Megacariócitos/metabolismo , Mielofibrose Primária/patologia , Receptor 4 Toll-Like/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Animais , Estudos de Casos e Controles , Diferenciação Celular , Feminino , Fibronectinas/genética , Humanos , Masculino , Megacariócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Osteomielite/metabolismo , Osteomielite/patologia , Mielofibrose Primária/metabolismo , Trombopoetina/genética , Trombopoetina/metabolismo , Receptor 4 Toll-Like/genéticaRESUMO
Platelet-type von Willebrand disease is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIbα-von Willebrand factor (vWF) interaction. GPIbα and vWF play a role in megakaryocytopoiesis, thus we aimed to investigate megakaryocyte differentiation and proplatelet-formation in platelet-type von Willebrand disease using megakaryocytes from a patient carrying the Met239Val variant and from mice carrying the Gly233Val variant. Platelet-type von Willebrand disease megakaryocytes bound vWF at an early differentiation stage and generated proplatelets with a decreased number of enlarged tips compared to control megakaryocytes. Moreover, they formed proplatelets upon contact with collagen, differently from normal megakaryocytes. Similarly, collagen triggered megakaryocytes showed defective activation of the RhoA-MLC2 axis, which prevents proplatelet formation, and increased phosphorylation of Lyn, which acts as a negative regulator of GPVI signaling, thus preventing ectopic proplatelet-formation on collagen. Consistently, human and murine bone marrow contained an increased number of extravascular platelets compared to controls. In addition, platelet survival of mutant mice was shortened compared to control mice, and the administration of desmopressin, raising circulating vWF, caused a marked drop in platelet count. Taken together, these results show for the first time that thrombocytopenia in platelet-type von Willebrand disease is due to the combination of different pathogenic mechanisms, i.e. the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/vWF complexes.
Assuntos
Plaquetas/patologia , Megacariócitos/patologia , Mutação , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombocitopenia/fisiopatologia , Doenças de von Willebrand/patologia , Fator de von Willebrand/metabolismo , Animais , Plaquetas/metabolismo , Estudos de Casos e Controles , Movimento Celular , Humanos , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Trombocitopenia/metabolismo , Trombopoese , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/genéticaRESUMO
In Primary Myelofibrosis (PMF), megakaryocyte dysplasia/hyperplasia determines the release of inflammatory cytokines that, in turn, stimulate stromal cells and induce bone marrow fibrosis. The pathogenic mechanism and the cells responsible for progression to bone marrow fibrosis in PMF are not completely understood. This review article aims to provide an overview of the crucial role of megakaryocytes in myelofibrosis by discussing the role and the altered secretion of megakaryocyte-derived soluble factors, enzymes and extracellular matrices that are known to induce bone marrow fibrosis.
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Metalloproteinases (MMPs) are zinc-dependent endopeptidases that play essential roles as the mediator of matrix degradation and remodeling during organogenesis, wound healing and angiogenesis. Although MMPs were originally identified as matrixin proteases that act in the extracellular matrix, more recent research has identified members of the MMP family in unusual locations within the cells, exerting distinct functions in addition to their established role as extracellular proteases. During thrombopoiesis, megakaryocytes (Mks) sort MMPs to nascent platelets through pseudopodial-like structure known as proplatelets. Previous studies identified gelatinases, MMP-2 and MMP-9, as a novel regulator system of Mks and the platelet function. In this work we have exploited a sensitive immunoassay to detect and quantify multiple MMP proteins and their localization, in conditioned medium and sub-cellular fractions of primary human CD34âº-derived Mks. We provide evidence that Mks express other MMPs in addition to gelatinases MMP-2 and MMP-9, peculiar isoforms of MMP-9 and MMPs with a novel nuclear compartmentalization.
Assuntos
Células Sanguíneas/fisiologia , Medula Óssea/efeitos dos fármacos , Fibronectinas/genética , Processamento Alternativo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Medula Óssea/fisiologia , Fibronectinas/fisiologia , Humanos , Camundongos , Domínios Proteicos , Recuperação de Função Fisiológica , RegeneraçãoRESUMO
Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic ß cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca2+ increase ([Ca2+] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 µm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner.
Assuntos
Ácido Abscísico/farmacologia , Megacariócitos/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Trombopoese/efeitos dos fármacos , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Fosfato , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Superfície Celular/metabolismo , Trombopoetina/metabolismoRESUMO
Patients with inflammatory bowel disease (IBD) are at higher risk of venous thromboembolism and coronary artery disease despite having a lower burden of traditional risk factors. Platelets from IBD patients release more soluble CD40 ligand (CD40L), and this has been implicated in IBD platelet hyper-activation. We here measured the urinary F2-isoprostane 8-iso-prostaglandin (PG)2α (8-iso-PGF2α), urinary 11-dehydro-thromboxane (TX) B2 (11-dehydro-TXB2) and plasma CD40L in IBD patients, and explored the in vitro action of anti-tumour necrosis factor (TNF)-α antibody infliximab on IBD differentiating megakaryocytes. Urinary and blood samples were collected from 124 IBD patients and 37 healthy subjects. Thirteen IBD patients were also evaluated before and after 6-week infliximab treatment. The in vitro effect of infliximab on patient-derived megakaryocytes was evaluated by immunoflorescence microscopy and by flow cytometry. IBD patients had significantly (p<0.0001) higher urinary 8-iso-PGF2α and 11-dehydro-TXB2 as well as plasma CD40L levels than controls, with active IBD patients displaying higher urinary and plasma values when compared to inactive patients in remission. A 6-week treatment with infliximab was associated with a significant reduction of the urinary excretion of 8-iso-PGF2α and 11-dehydro-TXB2 (p=0.008) and plasma CD40L (p=0.001). Infliximab induced significantly rescued pro-platelet formation by megakaryocytes derived from IBD patients but not from healthy controls. Our findings provide evidence for enhanced in vivo TX-dependent platelet activation and lipid peroxidation in IBD patients. Anti-TNF-α therapy with infliximab down-regulates in vivo isoprostane generation and TX biosynthesis in responder IBD patients. Further studies are needed to clarify the implication of infliximab induced-proplatelet formation from IBD megakaryocytes.
Assuntos
Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab/uso terapêutico , Ativação Plaquetária , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Aterosclerose/prevenção & controle , Ligante de CD40/sangue , Colite Ulcerativa/sangue , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/sangue , Doença de Crohn/tratamento farmacológico , Feminino , Fármacos Gastrointestinais/uso terapêutico , Humanos , Técnicas In Vitro , Doenças Inflamatórias Intestinais/complicações , Peroxidação de Lipídeos , Masculino , Megacariócitos/efeitos dos fármacos , Megacariócitos/patologia , Pessoa de Meia-Idade , Estresse Oxidativo , Ativação Plaquetária/efeitos dos fármacos , Tromboxanos/metabolismo , Adulto JovemRESUMO
Fibronectin (FN) is a major extracellular matrix protein implicated in cell adhesion and differentiation in the bone marrow (BM) environment. Alternative splicing of FN gene results in the generation of protein variants containing an additional EIIIA domain that sustains cell proliferation or differentiation during physiological or pathological tissue remodeling. To date its expression and role in adult hematopoiesis has not been explored. In our research, we demonstrate that during physiological hematopoiesis a small fraction of BM derived FN contains the EIIIA domain and that mice constitutively including (EIIIA(+/+) ) or excluding (EIIIA(-/-) ) the EIIIA exon present comparable levels of hematopoietic stem cells, myeloid and lymphoid progenitors within BM. Moreover, only minor alterations were detected in blood parameters and in hematopoietic frequencies of BM granulocytes/monocytes and B cells. As opposed to other tissues, unique compensatory mechanisms, such as increased FN accumulation and variable expression of the EIIIA receptors, Toll like receptor-4 and alpha9 integrin subunit, characterized the BM of these mice. Our data demonstrate that FN is a fundamental component of the hematopoietic tissue and that the EIIIA exon may play a key role in modulating hematopiesis in conditions of BM stress or diseases. Stem Cells 2016;34:2263-2268.