Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
1.
Haematologica ; 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-38058210

RESUMO

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab-prophylaxis led us employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We thus analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab compared to rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab compared to rFVIII/rFVIIIFc. Scanning-electron microscopy and immunofluorescent spinning-disk confocal-microscopy of in vivo generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFc- and emicizumab-induced clots. Contrary to the regular fibrin-mesh obtained with rFVIII/rFVIIIFc, fibrin-fibers appeared to be fused into large patches upon emicizumabtreatment. Moreover, fewer red blood cells were detected in regions where these fibrin patches were present. The presence of highly-dense fibrin-structures associated with a diffuse fiber-structure in emicizumab-induced clots was also observed when using superresolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

2.
Breast Cancer Res ; 24(1): 39, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35659040

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.


Assuntos
Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Linfócitos T , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Microambiente Tumoral
3.
Blood ; 136(6): 740-748, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32369559

RESUMO

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator IX/administração & dosagem , Fator X/administração & dosagem , Hemofilia A/tratamento farmacológico , Hemorragia/tratamento farmacológico , Modelos Animais , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/imunologia , Quimioterapia Combinada , Fator IX/análise , Fator IX/imunologia , Fator VIII/administração & dosagem , Fator VIII/análise , Fator VIII/uso terapêutico , Fator X/análise , Fator X/imunologia , Fator XIa/farmacologia , Feminino , Hemofilia A/sangue , Hemofilia A/complicações , Hemofilia A/imunologia , Hemorragia/etiologia , Infusões Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tempo de Tromboplastina Parcial , Cauda/lesões , Trombina/biossíntese
4.
Blood ; 132(11): 1193-1197, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30064978

RESUMO

Von Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip-bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 µL vs 194 ± 146 µL; P = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII.


Assuntos
Formação de Anticorpos/efeitos dos fármacos , Autoanticorpos , Fator VIII , Proteínas Recombinantes de Fusão , Anticorpos de Domínio Único/farmacologia , Fator de von Willebrand , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Fator VIII/imunologia , Fator VIII/farmacocinética , Fator VIII/farmacologia , Camundongos , Camundongos Mutantes , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Fator de von Willebrand/imunologia , Fator de von Willebrand/metabolismo
5.
Blood ; 129(17): 2443-2454, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28213380

RESUMO

Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 µM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K-dependent proteins was observed. Short hairpin RNA-mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI-deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti-vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI-mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.


Assuntos
Proteína C-Reativa/metabolismo , Deficiência do Fator X/sangue , Fator X/metabolismo , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Depuradores Classe A/metabolismo , Animais , Anticoagulantes/farmacologia , Proteína C-Reativa/genética , Linhagem Celular , Endocitose , Fator X/genética , Deficiência do Fator X/genética , Deficiência do Fator X/patologia , Expressão Gênica , Células HEK293 , Humanos , Cinética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Depuradores Classe A/antagonistas & inibidores , Receptores Depuradores Classe A/deficiência , Receptores Depuradores Classe A/genética , Vitamina K/antagonistas & inibidores , Vitamina K/metabolismo
6.
Blood ; 127(6): 778-86, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26608330

RESUMO

Beside its classical role in the coagulation cascade, coagulation factor X (FX) is involved in several major biological processes including inflammation and enhancement of virus-induced immune responses. We recently reported that the long circulatory half-life of FX is linked to its interaction with liver-resident macrophages. Importantly, we now observed that macrophages, but not undifferentiated monocytes, support this interaction. Using cell biology approaches with primary and THP1-derived macrophages as well as transfected cells, we further identified the scavenger receptor type A member I (SR-AI) to be a macrophage-specific receptor for FX. This result was confirmed using SR-AI-deficient mice, which exhibit reduced circulating levels of FX in vivo and loss of FX-macrophage interactions in vitro. Binding studies using purified proteins revealed that FX binds specifically (half-maximal binding, 3 µg/mL) to the extracellular domain of SR-AI. Altogether, we demonstrate that macrophages regulate FX plasma levels in an SR-AI-dependent manner.


Assuntos
Fator X/metabolismo , Receptores Depuradores Classe A/fisiologia , Animais , Coagulação Sanguínea/genética , Diferenciação Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/metabolismo , Monócitos/fisiologia , Receptores Depuradores Classe A/genética
7.
Haematologica ; 103(4): 728-737, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29326120

RESUMO

Previously, we found that LDL-receptor related protein-1 on macrophages mediated shear stress-dependent clearance of von Willebrand factor. In control experiments, however, we observed that von Willebrand factor also binds to macrophages independently of this receptor under static conditions, suggesting the existence of additional clearance-receptors. In search for such receptors, we focused on the macrophage-specific scavenger-receptor SR-AI. von Willebrand factor displays efficient binding to SR-AI (half-maximum binding 14±5 nM). Binding is calcium-dependent and is inhibited by 72±4% in the combined presence of antibodies against the A1- and D4-domains. Association with SR-AI was confirmed in cell-binding experiments. In addition, binding to bone marrow-derived murine SR-AI-deficient macrophages was strongly reduced compared to binding to wild-type murine macrophages. Following expression via hydrodynamic gene transfer, we determined ratios for von Willebrand factor-propeptide over von Willebrand factor-antigen, a marker of von Willebrand factor clearance. Propeptide/antigen ratios were significantly reduced in SR-AI-deficient mice compared to wild-type mice (0.6±0.2 versus 1.3±0.3; P<0.0001), compatible with a slower clearance of von Willebrand factor in SR-AI-deficient mice. Interestingly, mutants associated with increased clearance (von Willebrand factor/p.R1205H and von Willebrand factor/p.S2179F) had significantly increased binding to purified SR-AI and SR-AI expressed on macrophages. Accordingly, propeptide/antigen ratios for these mutants were reduced in SR-AI-deficient mice. In conclusion, we have identified SR-AI as a novel macrophage-specific receptor for von Willebrand factor. Enhanced binding of von Willebrand factor mutants to SR-AI may contribute to the increased clearance of these mutants.


Assuntos
Receptores Depuradores Classe A/fisiologia , Fator de von Willebrand/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Macrófagos , Camundongos , Proteínas Mutantes/metabolismo , Ligação Proteica , Receptores Depuradores/fisiologia , Fator de von Willebrand/genética
8.
Mol Hum Reprod ; 20(10): 960-71, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25082981

RESUMO

We identified three doublesex and mab-3-related transcription factors (DMRT) that were sexually differentially expressed in human fetal gonads and present in the ovaries at the time of meiotic initiation. These were also identified in murine embryonic female germ cells. Among these, we focused on DMRTA2 (DMRT5), whose function is unknown in the developing gonads, and clarified its role in human female fetal germ cells, using an original xenograft model. Early human fetal ovaries (8-11 weeks post-fertilization) were grafted into nude mice. Grafted ovaries developed normally, with no apparent overt changes, when compared with ungrafted ovaries at equivalent developmental stages. Appropriate germ cell density, mitotic/meiotic transition, markers of meiotic progression and follicle formation were evident. Four weeks after grafting, mice were treated with siRNA, specifically targeting human DMRTA2 mRNA. DMRTA2 inhibition triggered an increase in undifferentiated FUT4-positive germ cells and a decrease in the percentage of meiotic γH2AX-positive germ cells, when compared with mice that were injected with control siRNA. Interestingly, the expression of markers associated with pre-meiotic germ cell differentiation was also impaired, as was the expression of DMRTB1 (DMRT6) and DMRTC2 (DMRT7). This study reveals, for the first time, the requirement of DMRTA2 for normal human female embryonic germ cell development. DMRTA2 appears to be necessary for proper differentiation of oogonia, prior to entry into meiosis, in the human species. Additionally, we developed a new model of organ xenografting, coupled with RNA interference, which provides a useful tool for genetic investigations of human germline development.


Assuntos
Fucosiltransferases/metabolismo , Histonas/metabolismo , Antígenos CD15/metabolismo , Ovário/transplante , Óvulo/citologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos SCID , Ovário/crescimento & desenvolvimento , Óvulo/crescimento & desenvolvimento , Interferência de RNA , RNA Interferente Pequeno , Fatores de Transcrição/biossíntese , Transplante Heterólogo
9.
Reproduction ; 147(4): R119-29, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24497529

RESUMO

Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.


Assuntos
Experimentação Animal/normas , Disruptores Endócrinos/toxicidade , Roedores , Testes de Toxicidade/normas , Animais , Humanos , Masculino , Camundongos , Modelos Animais , Ratos , Medição de Risco , Testículo/efeitos dos fármacos , Testes de Toxicidade/métodos
10.
Thromb Res ; 236: 242-249, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38383218

RESUMO

Early gene therapy clinical trials for the treatment of Haemophilia B have been instrumental to our global understanding of gene therapy and have significantly contributed to the rapid expansion of the field. The use of adeno-associated viruses (AAVs) as vectors for gene transfer has successfully led to therapeutic expression of coagulation factor IX (FIX) in severe haemophilia B patients. Expression of FIX has remained stable following a single administration of vector for up to 8 years at levels that are clinically relevant to reduce the incidence of spontaneous bleeds and have permitted a significant change in the disease management with reduction or elimination of the need for coagulation factor concentrates. These trials have also shed light on several concerns around AAV-mediated gene transfer such as the high prevalence of pre-existing immunity against the vector capsid as well as the elevation of liver transaminases that is associated with a loss of FIX transgene expression in some patients. However, this field is advancing very rapidly with the development of increasingly more efficient strategies to overcome some of these obstacles and importantly raise the possibility of a functional cure, which has been long sought after. This review overviews the evolution of gene therapy for haemophilia B over the last two decades.


Assuntos
Hemofilia B , Humanos , Hemofilia B/genética , Hemofilia B/terapia , Vetores Genéticos , Terapia Genética , Fator IX/genética , Fator IX/uso terapêutico , Fator IX/metabolismo , Hemorragia/tratamento farmacológico , Dependovirus/genética , Dependovirus/metabolismo
12.
J Thromb Haemost ; 20(3): 755-766, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34816579

RESUMO

BACKGROUND: Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. OBJECTIVES: To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain. METHODS: The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF. RESULTS: Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma. CONCLUSIONS: Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.


Assuntos
Doenças de von Willebrand , Fator de von Willebrand , Animais , Plaquetas/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , Osteoprotegerina/metabolismo , Adesividade Plaquetária , Ristocetina , Doenças de von Willebrand/genética , Fator de von Willebrand/metabolismo
13.
Cardiovasc Res ; 118(2): 622-637, 2022 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33576766

RESUMO

AIMS: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. METHODS AND RESULTS: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvß3 signalling abolished proliferation. However, VWF did not bind to αvß3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvß3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. CONCLUSION: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvß3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.


Assuntos
Aterosclerose/metabolismo , Proliferação de Células , Integrina alfaVbeta3/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de von Willebrand/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Movimento Celular , Células Cultivadas , Hiperplasia , Integrina alfaVbeta3/genética , Proteínas Relacionadas a Receptor de LDL/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Placa Aterosclerótica , Transdução de Sinais , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Fator de von Willebrand/genética
15.
Environ Health Perspect ; 117(1): 32-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19165384

RESUMO

BACKGROUND: Several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat. OBJECTIVES: Using the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate--mono-2-ethylhexyl phthalate (MEHP)--an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function. METHODS: Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH). RESULTS: Whatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10(-4) M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10(-4) M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3-positive germ cells, without modification of their proliferation. CONCLUSIONS: This is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.


Assuntos
Células Germinativas/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Testículo/efeitos dos fármacos , Testosterona/biossíntese , Células Germinativas/citologia , Humanos , Masculino , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/citologia , Testículo/embriologia , Testículo/metabolismo
16.
Thromb Haemost ; 119(12): 1981-1993, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31639831

RESUMO

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.


Assuntos
Fator X/genética , Hemofilia A/genética , Hemofilia A/terapia , Hemostasia , Trombina/química , Animais , Anticorpos/química , Modelos Animais de Doenças , Feminino , Fibrinopeptídeo A/genética , Variação Genética , Hepatócitos/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Peptídeos , Domínios Proteicos , Proteínas Recombinantes/química
17.
Sci Rep ; 6: 37953, 2016 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-27892504

RESUMO

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Inflamação/patologia , Lectinas/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Glicoproteínas de Membrana/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antígenos CD/genética , Antígenos CD/farmacologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/farmacologia , Modelos Animais de Doenças , Selectina E/metabolismo , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lectinas/genética , Lectinas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , Domínios e Motivos de Interação entre Proteínas , Solubilidade , Fator de Necrose Tumoral alfa/toxicidade
18.
JCI Insight ; 1(16): e88643, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27734030

RESUMO

von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.


Assuntos
Fatores de Despolimerização de Actina/genética , Quinases Lim/genética , Trombocitopenia/fisiopatologia , Doença de von Willebrand Tipo 2/fisiopatologia , Fator de von Willebrand/genética , Animais , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Mutação , Transdução de Sinais , Proteínas rho de Ligação ao GTP , Proteína rhoA de Ligação ao GTP , Doença de von Willebrand Tipo 2/enzimologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-25999913

RESUMO

During the last decades, many studies reported that male reproductive disorders are increasing among humans. It is currently acknowledged that these abnormalities can result from fetal exposure to environmental chemicals that are progressively becoming more concentrated and widespread in our environment. Among the chemicals present in the environment (air, water, food, and many consumer products), several can act as endocrine disrupting compounds (EDCs), thus interfering with the endocrine system. Phthalates, bisphenol A (BPA), and diethylstilbestrol (DES) have been largely incriminated, particularly during the fetal and neonatal period, due to their estrogenic and/or anti-androgenic properties. Indeed, many epidemiological and experimental studies have highlighted their deleterious impact on fetal and neonatal testis development. As EDCs can affect many different genomic and non-genomic pathways, the mechanisms underlying the adverse effects of EDC exposure are difficult to elucidate. Using literature data and results from our laboratory, in the present review, we discuss the role of classical nuclear receptors (genomic pathway) in the fetal and neonatal testis response to EDC exposure, particularly to phthalates, BPA, and DES. Among the nuclear receptors, we focused on some of the most likely candidates, such as peroxisome-proliferator activated receptor (PPAR), androgen receptor (AR), estrogen receptors (ERα and ß), liver X receptors (LXR), and small heterodimer partner (SHP). First, we describe the expression and potential functions (based on data from studies using receptor agonists and mouse knockout models) of these nuclear receptors in the developing testis. Then, for each EDC studied, we summarize the main evidences indicating that the reprotoxic effect of each EDC under study is mediated through a specific nuclear receptor(s). We also point-out the involvement of other receptors and nuclear receptor-independent pathways.

20.
PLoS One ; 7(10): e48266, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23118965

RESUMO

BACKGROUND: Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. CONCLUSIONS/SIGNIFICANCE: We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.


Assuntos
Dietilexilftalato/análogos & derivados , Feto/metabolismo , Receptores Nucleares Órfãos/genética , Ovário/citologia , Ovário/metabolismo , Testículo/citologia , Testículo/metabolismo , Dietilexilftalato/toxicidade , Poluentes Ambientais/toxicidade , Feminino , Feto/citologia , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Lipídeos/biossíntese , Receptores X do Fígado , Masculino , Ovário/efeitos dos fármacos , Óvulo/citologia , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA