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1.
Proc Natl Acad Sci U S A ; 119(40): e2206515119, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36161923

RESUMO

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.


Assuntos
Infecções por Escherichia coli , Pielonefrite , Infecções Urinárias , Escherichia coli Uropatogênica , alfa-Defensinas , Animais , Variações do Número de Cópias de DNA , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Loci Gênicos , Humanos , Camundongos , Camundongos Transgênicos , Pielonefrite/genética , Pielonefrite/imunologia , Pielonefrite/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , alfa-Defensinas/genética
2.
Br J Haematol ; 196(4): 923-927, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34622440

RESUMO

Patients who are severely affected by coronavirus disease 2019 (COVID-19) may develop a delayed onset 'cytokine storm', which includes an increase in interleukin-6 (IL-6). This may be followed by a pro-thrombotic state and increased D-dimers. It was anticipated that tocilizumab (TCZ), an anti-IL-6 receptor monoclonal antibody, would mitigate inflammation and coagulation in patients with COVID-19. However, clinical trials with TCZ have recorded an increase in D-dimer levels. In contrast to TCZ, colchicine reduced D-dimer levels in patients with COVID-19. To understand how the two anti-inflammatory agents have diverse effects on D-dimer levels, we present data from two clinical trials that we performed. In the first trial, TCZ was administered (8 mg/kg) to patients who had a positive polymerase chain reaction test for COVID-19. In the second trial, colchicine was given (0·5 mg twice a day). We found that TCZ significantly increased IL-6, α-Defensin (α-Def), a pro-thrombotic peptide, and D-dimers. In contrast, treatment with colchicine reduced α-Def and Di-dimer levels. In vitro studies show that IL-6 stimulated the release of α-Def from human neutrophils but in contrast to colchicine, TCZ did not inhibit the stimulatory effect of IL-6; raising the possibility that the increase in IL-6 in patients with COVID-19 treated with TCZ triggers the release of α-Def, which promotes pro-thrombotic events reflected in an increase in D-dimer levels.


Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Tratamento Farmacológico da COVID-19 , Colchicina/uso terapêutico , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , alfa-Defensinas/imunologia , Idoso , Coagulação Sanguínea/efeitos dos fármacos , COVID-19/sangue , COVID-19/imunologia , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/imunologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/imunologia , Humanos , Interleucina-6/sangue , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia
3.
J Biol Chem ; 295(21): 7261-7273, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32280065

RESUMO

The integrin-binding secreted protein developmental endothelial locus-1 (DEL-1) is involved in the regulation of both the initiation and resolution of inflammation in different diseases, including periodontitis, an oral disorder characterized by inflammatory bone loss. Here, using a mouse model of bone regeneration and in vitro cell-based mechanistic studies, we investigated whether and how DEL-1 can promote alveolar bone regeneration during resolution of experimental periodontitis. Compared with WT mice, mice lacking DEL-1 or expressing a DEL-1 variant with an Asp-to-Glu substitution in the RGD motif ("RGE point mutant"), which does not interact with RGD-dependent integrins, exhibited defective bone regeneration. Local administration of DEL-1 or of its N-terminal segment containing the integrin-binding RGD motif, but not of the RGE point mutant, reversed the defective bone regeneration in the DEL-1-deficient mice. Moreover, DEL-1 (but not the RGE point mutant) promoted osteogenic differentiation of MC3T3-E1 osteoprogenitor cells or of primary calvarial osteoblastic cells in a ß3 integrin-dependent manner. The ability of DEL-1 to promote in vitro osteogenesis, indicated by induction of osteogenic genes such as the master transcription factor Runt-related transcription factor-2 (Runx2) and by mineralized nodule formation, depended on its capacity to induce the phosphorylation of focal adhesion kinase (FAK) and of extracellular signal-regulated kinase 1/2 (ERK1/2). We conclude that DEL-1 can activate a ß3 integrin-FAK-ERK1/2-RUNX2 pathway in osteoprogenitors and promote new bone formation in mice. These findings suggest that DEL-1 may be therapeutically exploited to restore bone lost due to periodontitis and perhaps other osteolytic conditions.


Assuntos
Regeneração Óssea , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Osteoblastos/metabolismo , Osteogênese , Animais , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoblastos/citologia
4.
Br J Haematol ; 194(1): 44-52, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34053084

RESUMO

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.


Assuntos
COVID-19/metabolismo , Inflamação/metabolismo , Tromboembolia/prevenção & controle , alfa-Defensinas/sangue , Adulto , Idoso , Animais , Coagulação Sanguínea/efeitos dos fármacos , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/virologia , Estudos de Casos e Controles , Colchicina/farmacologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Inflamação/complicações , Interleucina-6/sangue , Interleucina-6/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Neutrófilos/efeitos dos fármacos , Estudos Prospectivos , Fatores de Risco , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Tromboembolia/etiologia , Trombose/etiologia , Trombose/metabolismo , Moduladores de Tubulina/farmacologia , alfa-Defensinas/farmacologia
5.
Blood ; 133(5): 481-493, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30442678

RESUMO

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.


Assuntos
Fibrina/imunologia , Ativação de Neutrófilo , Trombose/imunologia , alfa-Defensinas/imunologia , Animais , Coagulação Sanguínea , Fibrina/análise , Fibrinólise , Humanos , Inflamação/sangue , Inflamação/imunologia , Inflamação/patologia , Calicreínas/sangue , Calicreínas/imunologia , Masculino , Camundongos , Conformação Proteica , Estabilidade Proteica , Trombose/sangue , Trombose/patologia , alfa-Defensinas/sangue
6.
Blood ; 128(1): 110-9, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27207796

RESUMO

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 µM and 45 µM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 µM), soluble VWF (KD = 0.58 µM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n = 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n = 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001). There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman ρ = 0.7932, P < .0001). Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders.


Assuntos
Proteína ADAMTS13/metabolismo , Defensinas/metabolismo , Neutrófilos/metabolismo , Proteólise , Púrpura Trombocitopênica Trombótica/metabolismo , Fator de von Willebrand/metabolismo , Motivos de Aminoácidos , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Neutrófilos/patologia , Púrpura Trombocitopênica Trombótica/patologia
7.
Am J Respir Cell Mol Biol ; 56(2): 261-270, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27755915

RESUMO

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7-/- and Cxcl4-/- knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7-/- mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4-/- mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.


Assuntos
Lesão Pulmonar Aguda/sangue , Plaquetas/metabolismo , Quimiocinas CXC/sangue , Fator Plaquetário 4/sangue , Animais , Permeabilidade Capilar , Humanos , Pulmão/irrigação sanguínea , Pulmão/patologia , Camundongos Transgênicos
8.
J Biol Chem ; 291(29): 15029-45, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27151212

RESUMO

Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(-/-)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(-/-) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(-/-) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis.


Assuntos
Proteínas de Homeodomínio/metabolismo , Neovascularização Fisiológica , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Células K562 , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
9.
Mol Psychiatry ; 20(7): 880-888, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25385367

RESUMO

Inflammation in the central nervous system (CNS) and disruption of its immune privilege are major contributors to the pathogenesis of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor, which inhibits integrin-dependent leukocyte adhesion. Here we show that Del-1 contributes to the immune privilege status of the CNS. Intriguingly, Del-1 expression decreased in chronic-active MS lesions and in the inflamed CNS in the course of EAE. Del-1-deficiency was associated with increased EAE severity, accompanied by increased demyelination and axonal loss. As compared with control mice, Del-1(-/-) mice displayed enhanced disruption of the blood-brain barrier and increased infiltration of neutrophil granulocytes in the spinal cord in the course of EAE, accompanied by elevated levels of inflammatory cytokines, including interleukin-17 (IL-17). The augmented levels of IL-17 in Del-1-deficiency derived predominantly from infiltrated CD8(+) T cells. Increased EAE severity and neutrophil infiltration because of Del-1-deficiency was reversed in mice lacking both Del-1 and IL-17 receptor, indicating a crucial role for the IL-17/neutrophil inflammatory axis in EAE pathogenesis in Del-1(-/-) mice. Strikingly, systemic administration of Del-1-Fc ameliorated clinical relapse in relapsing-remitting EAE. Therefore, Del-1 is an endogenous homeostatic factor in the CNS protecting from neuroinflammation and demyelination. Our findings provide mechanistic underpinnings for the previous implication of Del-1 as a candidate MS susceptibility gene and suggest that Del-1-centered therapeutic approaches may be beneficial in neuroinflammatory and demyelinating disorders.


Assuntos
Axônios/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte/metabolismo , Bainha de Mielina/metabolismo , Neuroimunomodulação/fisiologia , Medula Espinal/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proteínas de Ligação ao Cálcio , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Proteínas de Transporte/genética , Moléculas de Adesão Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Granulócitos/patologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Neuroimunomodulação/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Índice de Gravidade de Doença , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia
10.
Transfusion ; 56(7): 1775-85, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27040023

RESUMO

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.


Assuntos
Proteína ADAMTS13/antagonistas & inibidores , Autoanticorpos , Púrpura Trombocitopênica Trombótica/imunologia , Púrpura Trombocitopênica Trombótica/terapia , Proteína ADAMTS13/imunologia , Animais , Autoanticorpos/genética , Clonagem Molecular , DNA Complementar/administração & dosagem , Humanos , Camundongos , Modelos Animais , Terapia de Alvo Molecular/métodos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/toxicidade , Fator de von Willebrand/metabolismo
11.
Circ Res ; 110(9): 1202-10, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22456181

RESUMO

RATIONALE: Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. OBJECTIVE: We addressed the role of platelets in mediating CNS inflammation in EAE. METHODS AND RESULTS: We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control nondiseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression on platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ibα (GPIbα) promotes both platelet adhesion and inflammatory actions of platelets and targeting of GPIbα attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), also reduced EAE severity in mice. CONCLUSIONS: Platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.


Assuntos
Plaquetas/metabolismo , Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/sangue , Leucócitos/imunologia , Animais , Anti-Inflamatórios/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Células Cultivadas , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/sangue , Camundongos , Camundongos Endogâmicos C57BL , Adesividade Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fatores de Tempo
12.
Blood Adv ; 8(14): 3798-3809, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38805575

RESUMO

ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.


Assuntos
Megacariócitos , Ativador de Plasminogênio Tipo Uroquinase , Megacariócitos/metabolismo , Megacariócitos/citologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Humanos , Animais , Camundongos , Fibrinólise/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Plaquetas/metabolismo , Trombose/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Grânulos Citoplasmáticos/metabolismo , Antígenos CD34/metabolismo
13.
bioRxiv ; 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38106191

RESUMO

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points: Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbß3 receptors and is functionally available from activated platelets.

14.
J Thromb Haemost ; 21(3): 652-666, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36696211

RESUMO

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. OBJECTIVES: To determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti-PF4 antibody (hRTO). METHODS: Results: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. CONCLUSIONS: HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.


Assuntos
Trombocitopenia , Trombose , Humanos , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticoagulantes/efeitos adversos , Complexo Antígeno-Anticorpo , Fondaparinux/efeitos adversos , Heparina/efeitos adversos , Fator Plaquetário 4 , Receptores de IgG , Trombose/etiologia
15.
Am J Respir Cell Mol Biol ; 46(2): 196-206, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22298529

RESUMO

The low-density lipoprotein receptor-related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, collagen synthesis, proteolysis, and migration in pleural mesothelial cells (PMCs). When PMCs were treated with the proinflammatory cytokines TNF-α and IL-1ß, LRP-1 significantly decreased at the mRNA and protein levels (70 and 90%, respectively; P < 0.05). Consequently, uPA-mediated uPAR internalization was reduced by 80% in the presence of TNF-α or IL-1ß (P < 0.05). In parallel studies, LRP-1 neutralization with receptor-associated protein (RAP) significantly reduced uPA-dependent uPAR internalization and increased uPAR stability in PMCs. LRP-1-deficient cells demonstrated increased uPAR t(1/2) versus LRP-1-expressing PMCs. uPA enzymatic activity was also increased in LRP-1-deficient and neutralized cells, and RAP potentiated uPA-dependent migration in PMCs. Collagen expression in PMCs was also induced by uPA, and the effect was potentiated in RAP-treated cells. These studies indicate that TNF-α and IL-1ß regulate LRP-1 in PMCs and that LRP-1 thereby contributes to a range of pathophysiologically relevant responses of these cells.


Assuntos
Colágeno Tipo I/metabolismo , Epitélio/metabolismo , Pleura/metabolismo , Receptores de Lipoproteínas/metabolismo , Linhagem Celular , Humanos , Pleura/citologia , Proteólise
16.
J Biol Chem ; 286(26): 23044-53, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21540184

RESUMO

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of ß-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.


Assuntos
Barreira Alveolocapilar/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Mucosa Respiratória/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Barreira Alveolocapilar/patologia , Permeabilidade Capilar/genética , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Edema Pulmonar/genética , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Mucosa Respiratória/patologia , Serpina E2/genética , Serpina E2/metabolismo , Serpina E2/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
17.
Am J Physiol Lung Cell Mol Physiol ; 301(3): L307-14, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21571905

RESUMO

Tissue-type plasminogen activator (tPA) is a potent fibrinolytic enzyme used to treat acute coronary artery obstruction. However, tPA has shown limited utility in other disorders caused by thrombotic vascular occlusion, such as pulmonary embolism. We found that tPA caused dose-dependent effects on the contractility of pulmonary arterial rings that may affect its effectiveness as a thrombolytic agent. At low concentrations (1 nM), tPA stimulated pulmonary vascular contraction in response to phenylephrine, whereas at higher concentrations (20 nM) tPA inhibited pulmonary arterial contractility and promoted pulmonary vascular permeability through an interaction between its "docking site" and N-methyl d-aspartate receptor type 1 (NMDA-R1) expressed by pulmonary arteries. A hexapeptide derived from plasminogen activator inhibitor type 1 that blocked the docking site of tPA, but not its catalytic activity, inhibited its interaction with NMDA-R1, abolished inhibition of pulmonary artery contractility, attenuated vascular permeability, and facilitated fibrinolysis in a murine model of pulmonary embolism. Similar outcomes were seen using a tPA variant that lacks the docking site but retains catalytic activity. These data suggest that it is feasible to attenuate the deleterious extrafibrinolytic effects of tPA and improve its benefit:risk profile in the management of pulmonary embolism.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/fisiologia , Ativador de Plasminogênio Tecidual/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Endotélio Vascular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenilefrina/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Embolia Pulmonar , Ratos , Ratos Sprague-Dawley , Ativador de Plasminogênio Tecidual/administração & dosagem
18.
Blood ; 114(11): 2290-8, 2009 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-19605848

RESUMO

Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis, but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells, is maximally present on large, polyploidy megakaryocytes, and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP), an antagonist of LDL family member receptors, or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition, using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4(High)). Further, shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally, infusion of RAP into hPF4(High) mice was able to increase baseline platelet counts without affecting other lineages, suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1, which is transiently expressed during megakaryopoiesis.


Assuntos
Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Comunicação Parácrina/fisiologia , Fator Plaquetário 4/metabolismo , Receptores de LDL/metabolismo , Trombopoese/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Anticorpos/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/genética , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Comunicação Parácrina/efeitos dos fármacos , Fator Plaquetário 4/genética , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/genética , Trombopoese/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética
19.
Am J Respir Crit Care Med ; 182(12): 1516-23, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20656938

RESUMO

RATIONALE: Phagocytosis of apoptotic cells, also called efferocytosis, plays an essential role in the resolution of inflammation. Urokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in inflammatory conditions, including pneumonia and severe infection, which are often accompanied by the development of acute lung injury. However, the role of uPA in modulating efferocytosis of apoptotic neutrophils has not been defined. OBJECTIVES: To characterize the role of uPA in regulation of efferocytosis and to delineate the underlying mechanisms involved in this process. METHODS: In vitro and in vivo phagocytosis, immunoprecipitation, and Western blotting assays. MEASUREMENTS AND MAIN RESULTS: The phagocytosis of apoptotic neutrophils by macrophages was significantly inhibited by uPA. Mutant uPA lacking the growth factor domain and catalytically inactive uPA had similar inhibitory effects on efferocytosis, as did wild-type uPA. In contrast, absence of the kringle domain abrogated the ability of uPA to diminish efferocytosis. Both the α(V)ß3 integrin and vitronectin seemed to be involved in the inhibition of efferocytosis by uPA. Incubation of macrophages with uPA also diminished activation of the small GTPase Rac-1, which normally occurs during ingestion of apoptotic neutrophils. Under in vivo conditions in the lungs, uPA decreased the uptake of apoptotic neutrophils by alveolar macrophages. CONCLUSIONS: Our data demonstrate a novel role for uPA in which it is able to diminish the uptake of apoptotic neutrophils by macrophages under both in vitro and in vivo conditions.


Assuntos
Lesão Pulmonar Aguda/patologia , Apoptose/efeitos dos fármacos , Neutrófilos/patologia , Fagocitose/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL
20.
Am J Respir Crit Care Med ; 181(9): 935-46, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20093642

RESUMO

RATIONALE: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. alpha-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species. OBJECTIVES: To study the role of alpha-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of alpha-defensins. METHODS: Transgenic mice expressing polymorphonuclear leukocyte alpha-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein-related receptor (LRP) in permeability was examined. MEASUREMENTS AND MAIN RESULTS: Acid aspiration induced neutrophil migration and release of alpha-defensins into lung parenchyma and airways. ALI was more severe in alpha-defensin-expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, alpha-defensin-expressing mice showed greater disruption of capillary-epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP. CONCLUSIONS: alpha-Defensins mediate ALI through LRP-mediated loss of capillary-epithelial barrier function, suggesting a potential new approach to intervention.


Assuntos
Lesão Pulmonar Aguda/fisiopatologia , alfa-Defensinas/fisiologia , Lesão Pulmonar Aguda/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Capilares/fisiologia , Permeabilidade Capilar , Células Epiteliais/fisiologia , Técnicas de Transferência de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutrófilos/química , alfa-Defensinas/metabolismo
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