RESUMO
Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.
Assuntos
Hemostáticos , Doenças de von Willebrand , Humanos , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Células Endoteliais/metabolismo , Hemostáticos/metabolismo , Corpos de Weibel-Palade/genética , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Doenças de von Willebrand/genética , Doenças de von Willebrand/metabolismo , Doenças de von Willebrand/patologiaRESUMO
Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17ß-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERß agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.
Assuntos
Células Endoteliais/efeitos dos fármacos , Estradiol/efeitos adversos , Exocitose/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Terapia de Reposição de Estrogênios/efeitos adversos , Exocitose/fisiologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/fisiologia , Fatores de Risco , Tromboembolia/etiologia , Corpos de Weibel-Palade/efeitos dos fármacos , Corpos de Weibel-Palade/patologia , Corpos de Weibel-Palade/fisiologiaRESUMO
Agonist-mediated exocytosis of Weibel-Palade bodies underpins the endothelium's ability to respond to injury or infection. Much of this important response is mediated by the major constituent of Weibel-Palade bodies: the ultra-large glycoprotein von Willebrand factor. Upon regulated WPB exocytosis, von Willebrand factor multimers unfurl into long, platelet-catching 'strings' which instigate the pro-haemostatic response. Accordingly, excessive levels of VWF are associated with thrombotic pathologies, including myocardial infarction and ischaemic stroke. Failure to appropriately cleave von Willebrand Factor strings results in thrombotic thrombocytopenic purpura, a life-threatening pathology characterised by tissue ischaemia and multiple microvascular occlusions. Historically, treatment of thrombotic thrombocytopenic purpura has relied heavily on plasma exchange therapy. However, the demonstrated efficacy of Rituximab and Caplacizumab in the treatment of acquired thrombotic thrombocytopenic purpura highlights how insights into pathophysiology can improve treatment options for von Willebrand factor-related disease. Directly limiting von Willebrand factor release from Weibel-Palade bodies has the potential as a therapeutic for cardiovascular disease. Cell biologists aim to map the WPB biogenesis and secretory pathways in order to find novel ways to control von Willebrand factor release. Emerging paradigms include the modulation of Weibel-Palade body size, trafficking and mechanism of fusion. This review focuses on the promise, progress and challenges of targeting Weibel-Palade bodies as a means to inhibit von Willebrand factor release from endothelial cells.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Fibrinolíticos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Púrpura Trombocitopênica Trombótica/tratamento farmacológico , Corpos de Weibel-Palade/efeitos dos fármacos , Fator de von Willebrand/antagonistas & inibidores , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/patologia , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Hemostasia/efeitos dos fármacos , Hemostasia/genética , Humanos , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Tamanho das Organelas/efeitos dos fármacos , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/metabolismo , Púrpura Trombocitopênica Trombótica/patologia , Rituximab/uso terapêutico , Via Secretória/efeitos dos fármacos , Via Secretória/genética , Anticorpos de Domínio Único/uso terapêutico , Corpos de Weibel-Palade/genética , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genéticaRESUMO
BACKGROUND: It is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5-5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. OBJECTIVE: We hypothesized that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. METHODS: Using a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. RESULTS AND CONCLUSIONS: A novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro-hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS-CoV-2 pandemic, with a considerable fraction of critically ill COVID-19 patients affected by hypercoagulability, these WPB size-reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.
Assuntos
Fibrinolíticos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Tamanho das Organelas/efeitos dos fármacos , Corpos de Weibel-Palade/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Hemostasia/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 ß1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 ß1, also the µ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.
Assuntos
Complexo 3 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Células Endoteliais , Exocitose , Síndrome de Hermanski-Pudlak , Proteínas R-SNARE/metabolismo , Corpos de Weibel-Palade , Complexo 3 de Proteínas Adaptadoras/genética , Complexo 3 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Sinalização do Cálcio , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismo , Síndrome de Hermanski-Pudlak/patologia , Humanos , Mutação , Transporte Proteico , Proteínas R-SNARE/genética , Corpos de Weibel-Palade/genética , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologiaRESUMO
The use of propranolol for the treatment of infantile hemangioma (IH) has been widely investigated in recent years. However, the underlying therapeutic mechanism of propranolol for the treatment of IH remains poorly understood. The aim of the present study was to investigate the expression of proteins regulated by cellular tumor antigen p53 (p53) in associated apoptosis pathways in IH endothelial cells (HemECs) treated with propranolol. Furthermore, the present study aimed to investigate the exact apoptotic pathway underlying the therapeutic effect of propranolol against IH. In the present study, HemECs were subcultured and investigated using an inverted phase contrast microscope, immunocytochemical staining and a scanning electron microscope (SEM). Experimental groups and blank control groups were prepared. All groups were subjected to drug treatment. A high p53 expression model of HemECs was successfully established via transfection, and a low p53 expression model of HemECs was established using pifithrinα. The apoptosis rate of each group was determined using Annexin Vfluorescein isothiocyanate/propidium iodide double staining and flow cytometry. The expression levels of downstream proteins regulated by p53 [tumour necrosis factor receptor superfamily member 6 (FAS), p53induced death domaincontaining protein (PIDD), death receptor 5 (DR5), BH3interacting domain death agonist (BID), apoptosis regulator BAX (BAX), p53 unregulated modulator of apoptosis (PUMA), phosphatidylinositolglycan biosynthesis class S protein (PIGS), and insulinlike growth factorbinding protein 3 (IGFBP3)] were revealed in the experimental and control groups via western blotting. Microscopic observation revealed the growth of an adherent monolayer of cells, which were closely packed and exhibited contact inhibition. Immunocytochemical staining demonstrated increased expression of clotting factor VIII. SEM analysis revealed presence of WeibelPalade bodies. The results of the analyses verified that the cultured cells were HemECs. The staining of the samples resulted in a significantly increased rate of apoptosis in experimental groups compared with the blank control group. This result suggested that there is an association between p53 expression and the rate of apoptosis of propranololtreated HemECs. The results of the western blot analysis demonstrated an upregulation of BAX expression and a downregulation of IGFBP3 expression in the HemECs treated with propranolol. There were no significant differences in the expression levels of FAS, DR5, PIDD, BID, PUMA and PIGS between experimental and control groups. This result suggests that p53 has an important role in HemEC apoptosis. The results of the present study additionally suggest that the propranololinduced HemEC apoptosis pathway is a mitochondrial apoptosis pathway and is regulated by p53BAX signaling.
Assuntos
Apoptose/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Hemangioma/metabolismo , Propranolol/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/genética , Células Endoteliais , Feminino , Hemangioma/genética , Hemangioma/patologia , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Propranolol/farmacologia , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Corpos de Weibel-Palade/genética , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Proteína X Associada a bcl-2/genéticaRESUMO
Background Circulating endothelial cells (CECs) are a potential biomarker of angiogenesis. CECs increase in numbers after vessel injury. Higher CEC numbers are reported in cancer patients. Most methods for CEC detection and enumeration rely on flow cytometry (FCM); however, there is no agreement on CEC phenotype and the detection method to be used. This leads to uncertainty about the clinical applicability and variation between studies on CEC numbers reported. Objective To develop a selective and accurate method for CEC enumeration in peripheral blood by enrichment, followed by FCM in healthy volunteers (HV) and cancer patients. Methods Samples were enriched using CD34 microbeads, stained with nuclear dye and anti-CD14, CD15, CD45, CD34 and CD146 antibodies. Putative CECs were examined for WeibelPalade bodies (WPBs) using antivon Willebrand factor (vWF) antibody and fluorescence microscopy. Linear range of detection (R 2), recovery and precision (coefficient of variation percentage [CV%]) were defined in three experiments by spiking a known number (range 1212,800 CECs/4 mL) of surrogate endothelial cells in peripheral blood. Sample storage was determined at 80°C for up to 2 months. Results Sorted CECs showed vWF in the WPBs. The relationship between spiked and detected surrogate cells was R 2 = 1.0, recovery of 94.0 to 101.4% and CV% of 1.0 to 18.4%. Recovery ± standard deviation (within-run days 1, 2 and 3) were, respectively, 102.5% ± 8.2, 97.8% ± 4.6, 99.1% ± 7.7, and after 2 months 94.3% ± 15.3. The median CECs/mL in patients was 24.1 versus 14.4 in HVs. Conclusion This method for selective, sensitive and reliable CEC analysis by FCM allows for investigation of CECs as a biomarker in clinical research.
Assuntos
Células Endoteliais/patologia , Citometria de Fluxo/métodos , Separação Imunomagnética/métodos , Corpos de Weibel-Palade/patologia , Adulto , Idoso , Antígenos CD34/metabolismo , Circulação Sanguínea , Células Endoteliais/metabolismo , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Países Baixos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: Retrospective studies suggest that donor desmopressin (DDAVP) treatment improves renal transplant outcome. The present study tests the hypothesis that desmopressin neutralizes the graft's endothelium from proinflammatory angiopoietin 2 containing Weibel-Palade bodies in the donor, resulting in reduced Weibel-Palade body release at the time of reperfusion in the recipient. MATERIALS AND METHODS: Using rat models, we examined the influence of desmopressin treatment on the expression of vasopressin 2 receptors and adhesion molecules in brain-dead donors, with renal function examined in allogeneic recipients. The influence of desmopressin on the expression of adhesion molecules also was tested in vitro. RESULTS: Vasopressin 2 receptors were restricted to collecting ducts and distal tubules and only scarcely found in the renal vasculature. Vasopressin 2 receptor expression was down-regulated in brain-dead rats by desmopressin. Renal expression of vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1 were significantly reduced in these rats. In contrast, angiopoietin 2 did not influence the expression of adhesion molecules in in vitro cultured endothelial cells after tumor necrosis factor ? stimulation. Donor desmopressin treatment improved neither renal function nor histology in allogeneic renal transplant recipients. CONCLUSIONS: Our data do not support the hypothesis that the clinically observed salutary effect of desmopressin is mediated by depletion of Weibel-Palade bodies in renal allografts.
Assuntos
Desamino Arginina Vasopressina/farmacologia , Células Endoteliais/efeitos dos fármacos , Transplante de Rim/efeitos adversos , Rim/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Corpos de Weibel-Palade/efeitos dos fármacos , Angiopoietina-2/farmacologia , Animais , Células Cultivadas , Isquemia Fria/efeitos adversos , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Modelos Animais , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Receptores de Vasopressinas/agonistas , Receptores de Vasopressinas/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fatores de Tempo , Transplante Homólogo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Isquemia Quente/efeitos adversos , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologiaRESUMO
In recent years, new functions for the haemostatic protein von Willebrand Factor (VWF) have emerged. Amongst these is the ability to modulate the development of new blood vessels, a process called angiogenesis. The subtle effects that VWF exerts on blood vessel formation and stability may be relevant for the small but significant fraction of patients with von Willebrand disease (VWD) who also present with vascular malformations (angiodysplasia) in the gastrointestinal tract, often responsible for intractable bleeding. This review will briefly summarise the evidence and discuss the molecular pathways involved.
Assuntos
Angiodisplasia/complicações , Angiodisplasia/patologia , Vasos Sanguíneos/patologia , Células Endoteliais/patologia , Doenças de von Willebrand/complicações , Doenças de von Willebrand/patologia , Fator de von Willebrand/metabolismo , Angiodisplasia/metabolismo , Animais , Trato Gastrointestinal/irrigação sanguínea , Humanos , Neovascularização Fisiológica , Transdução de Sinais , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Doenças de von Willebrand/metabolismoRESUMO
The large multimeric glyocoprotein von Willebrand factor (VWF) is a crucial component of both primary and secondary hemostasis. It is stored in secretory granules of vascular endothelial cells, the Weibel-Palade bodies (WPBs), and is released following stimulation by agonists that raise intracellular Ca(2+) or cyclic adenosine monophosphate (cAMP) levels. cAMP-induced exocytosis of WPBs requires protein kinase A activity, but downstream factors that are regulated by phosphorylation/dephosphorylation are not known. Here we identify the complex consisting of the lipid-binding protein annexin A2 (AnxA2) and S100A10 as such a factor. Knockdown and specific rescue approaches reveal that a functional AnxA2-S100A10 complex is required for the forskolin-induced, cAMP-dependent release of VWF. Forskolin triggers dephosphorylation of AnxA2 that is mediated by a calcineurin-like phosphatase and stabilizes the AnxA2-S100A10 complex, thereby promoting VWF release. Serine 11 of AnxA2 was identified as the target residue of this phosphorylation switch because a phosphomimicking mutation at this site prevents complex formation with S100A10 and, in contrast to wild-type or S11A-AnxA2, is unable to restore cAMP-dependent VWF secretion in AnxA2-depleted cells. Thus, complex formation of AnxA2 with S100A10 is a central regulatory mechanism in the acute release of VWF in response to cAMP-elevating agonists.
Assuntos
Anexina A2/metabolismo , AMP Cíclico/metabolismo , Endotélio/metabolismo , Fator de von Willebrand/metabolismo , Calcineurina/metabolismo , Cálcio/metabolismo , Colforsina/farmacologia , Exocitose , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas S100/metabolismo , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologiaRESUMO
Weibel-Palade bodies (WPBs) are the storage organelles for von Willebrand factor (VWF) in endothelial cells. VWF forms multimers that assemble into tubular structures in WPBs. Upon demand, VWF is secreted into the blood circulation, where it unfolds into strings that capture platelets during the onset of primary hemostasis. Numerous mutations affecting VWF lead to the bleeding disorder von Willebrand disease. This review reports the recent findings on the effects of VWF mutations on the biosynthetic pathway of VWF and its storage in WPBs. These new findings have deepened our understanding of VWF synthesis, storage, secretion, and function.
Assuntos
Corpos de Weibel-Palade/patologia , Doenças de von Willebrand/patologia , Humanos , Mutação , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genéticaRESUMO
BACKGROUND: von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. OBJECTIVES: We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel-Palade body (WPB)-like granules. METHODS: Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild-type VWF. Expression was also examined in HEK293 cells that form WPB-like granules when transfected with wild-type VWF. RESULTS: Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB-like granules, while R2663C was capable of WPB-like granules. Heterozygous expression of VWF variants had a negative impact on wild-type VWF with a reduction in elongated WPB-like granules in co-transfected cells. CONCLUSIONS: Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB-like granules.
Assuntos
Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Doença de von Willebrand Tipo 1/patologia , Fator de von Willebrand/metabolismo , Colágeno/metabolismo , Regulação para Baixo , Imunofluorescência , Predisposição Genética para Doença , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Microscopia Confocal , Mutação de Sentido Incorreto , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica , Multimerização Proteica , Transfecção , Doença de von Willebrand Tipo 1/sangue , Doença de von Willebrand Tipo 1/genética , Fator de von Willebrand/genéticaRESUMO
BACKGROUND: Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. DESIGN AND METHODS: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. RESULTS: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. CONCLUSIONS: We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.
Assuntos
Retículo Endoplasmático/genética , Mutação , Corpos de Weibel-Palade/genética , Fator de von Willebrand/genética , Cisteína/química , Cisteína/genética , Dissulfetos/química , Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Plasmídeos , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Serina/química , Serina/genética , Transfecção , Tirosina/química , Tirosina/genética , Corpos de Weibel-Palade/química , Corpos de Weibel-Palade/patologia , Doenças de von Willebrand/genética , Doenças de von Willebrand/metabolismo , Doenças de von Willebrand/patologia , Fator de von Willebrand/químicaRESUMO
Endothelial dysfunction is critical in the decline of renal function with. By using endothelial nitric oxide synthase knockout (eNOSKO) mice, we tested the hypothesis that a lack of endothelial nitric oxide synthase accelerates renal injury in the aging kidney. In contrast to control mice and young eNOSKO mice, aging eNOSKO mice showed greater renal injury and in particular developed a thrombotic microangiopathy, with mesangiolysis, endothelial swelling, endothelial cell loss, double-contour appearance of glomerular basement membrane (GBM), and thrombus formation. Thrombi, which were composed of fibrin, platelets, and von Willebrand factor (vWF), were identified predominantly in glomerular capillaries and rarely in arterioles, but not in larger vessels. In the tubulointerstitium, tubular degeneration and macrophage infiltration were also prominent in aging eNOSKO mice. Intraluminal vWF deposition was accompanied with thrombus formation, whereas mesangial deposition of vWF was associated with mesangial matrix expansion. Furthermore, the mesangial vWF deposition was detectable in young eNOSKO mice in which severe glomerular injury had not yet developed. Finally, a higher level of serum P-selectin in eNOSKO mice was consistent with the vWF behavior and suggested exocytosis of the Weibel-Palade body by the endothelium. In conclusion, a lack of endothelial nitric oxide synthase resulted in the development of glomerular thrombotic microangiopathy. A lack of nitric oxide likely contributed to the release of vWF, leading to thrombus formation in this model.
Assuntos
Envelhecimento , Endotélio Vascular/metabolismo , Regulação Enzimológica da Expressão Gênica , Rim/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Microangiopatias Trombóticas/metabolismo , Fator de von Willebrand/metabolismo , Animais , Exocitose , Masculino , Camundongos , Camundongos Knockout , Selectina-P/sangue , Trombose , Corpos de Weibel-Palade/patologiaRESUMO
Weibel-Palade bodies are uniquely present in endothelial cells and harbor a range of bioactive substances that participate in hemostasis, vasomotion, inflammation and fibrinolysis, in addition to modulating vascular permeability, angiogenic sprouting, and stem cell mobilization. This Perspectives article examines the latest insights into the biogenesis of these organelles and the cellular and molecular mechanisms of their exocytosis. In addition, we advance two hypotheses on the pathogenic role of these organelles: first, in the development of endothelial dysfunction associated with the reduction of nitric oxide bioavailability and accumulation of peroxynitrite and second, as a first-line response to acute stress that determines the balance between regenerative and proinflammatory signals.
Assuntos
Células Endoteliais/imunologia , Células Endoteliais/patologia , Estresse Fisiológico/imunologia , Corpos de Weibel-Palade/imunologia , Corpos de Weibel-Palade/patologia , Doença Aguda , Humanos , Inflamação/imunologia , Inflamação/patologiaRESUMO
OBJECT: Ultrastructural characteristics of hemorrhagic, nonhemorrhagic, primary, and recurrent central nervous system cavernous malformations (CMs) were examined in an attempt to clarify their pathological mechanisms. METHODS: Thirteen specimens (nine from samples of CMs and four from healthy control tissue) were processed for ultrastructural study immediately after surgical or postmortem removal, by fixation in glutaraldehyde/formalin and postfixation in OsO4. Transmission electron microscopy was used to examine the vascular walls, endothelium, subendothelium, and cytoplasmic organelles. The vascular walls in CMs demonstrated abnormal ultrastructure with no basement membranes and astrocytic foot processes. Pericytes were rarely seen. Single-layer lining endothelial cells showed fenestrated luminal surfaces. Large gaps were observed at intercellular junctions between endothelial cells, and large vesicles with extremely thin plasma membranes bordering the lumen were common in the lesions that had previously hemorrhaged. Endothelial cells of recurrent CMs had more Weibel-Palade bodies, filopodia, cytoplasmic processes, micropinocytotic vesicles, and filaments than those in primary lesions and normal control tissues. CONCLUSIONS: The absence of the blood-brain barrier, normal supporting wall structure, and large vesicles bordering the lumen of CM vessels may explain leakage of red blood cells into surrounding brain in the absence of major hemorrhage. Proliferation of residual abnormal endothelial cells may contribute to the recurrence of surgically removed CMs.
Assuntos
Neoplasias Encefálicas/patologia , Hemorragia Cerebral/patologia , Células Endoteliais/patologia , Hemangioma Cavernoso/patologia , Malformações Arteriovenosas Intracranianas/patologia , Adolescente , Adulto , Barreira Hematoencefálica , Neoplasias Encefálicas/cirurgia , Capilares/patologia , Capilares/ultraestrutura , Diferenciação Celular , Hemorragia Cerebral/cirurgia , Criança , Vesículas Citoplasmáticas/patologia , Células Endoteliais/ultraestrutura , Feminino , Hemangioma Cavernoso/cirurgia , Humanos , Malformações Arteriovenosas Intracranianas/cirurgia , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Pseudópodes/patologia , Recidiva , Vacúolos/patologia , Corpos de Weibel-Palade/patologiaRESUMO
Eight tumors diagnosed as solitary fibrous tumor (SFT) of the oral cavity were studied. Histologic spectrum was entirely comparable with the extrapleural SFT of other sites. One tumor had glomus tumor-like foci. Immunohistochemical results confirmed most of the previous observations, indicating characteristic expression of vimentin, CD34, bcl-2, and CD99. Factor XIIIa and alpha-smooth muscle actin were less commonly reactive and a very few cells were faintly positive for factor VIII-related antigen and Ulex europaeus agglutinin 1. All were essentially negative for S-100 protein, desmin, CD31, and CD68. In stark contrast to the conclusive immunoprofile, ultrastructural investigation of six tumors demonstrated considerable cellular heterogeneity. Other than fibroblasts, perivascular undifferentiated cells and pericytes predominated, but endothelial cells were regularly present. There was a distinctive proliferation of pericytic cells in four tumors, one of which had glomoid foci of myopericytes. The extreme increase in number of Weibel-Palade bodies occurred in voluminous capillary endothelium. Occasional single and clustered cells with consistent features of endothelium showed intracytoplasmic lumen formation. Such composite cells constituted an integral segment of richly vascularized SFT. Myofibroblastic form smooth muscle differentiation was present in only a minority of cells. From phenotypic analysis by electron microscopy, SFT may originate from a unique, perivascular multipotent mesenchyme sharing with its lineage with pericytes, fibroblasts, and infrequently, endothelium. Consequently, morphological features of SFT may become diversely varied by whether predominantly constituent cells are undifferentiated, pericytic or fibroblastic in nature.
Assuntos
Diferenciação Celular , Linhagem da Célula , Neoplasias Bucais/patologia , Neoplasias de Tecido Fibroso/patologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Feminino , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/ultraestrutura , Neoplasias de Tecido Fibroso/metabolismo , Neoplasias de Tecido Fibroso/ultraestrutura , Pericitos/patologia , Pericitos/ultraestrutura , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Corpos de Weibel-Palade/ultraestruturaRESUMO
Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD.
Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/patologia , Corpos de Weibel-Palade/metabolismo , Doenças de von Willebrand/metabolismo , Doenças de von Willebrand/patologia , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Sequência de Bases , Células Cultivadas , Cães , Mutação da Fase de Leitura/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Corpos de Weibel-Palade/genética , Corpos de Weibel-Palade/patologia , Doenças de von Willebrand/genética , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
BACKGROUND: Inducible nitric oxide synthase (iNOS, or NOS2) reduces the severity of accelerated graft arteriosclerosis (AGA) in transplanted organs, although the precise mechanism is unclear. METHODS AND RESULTS: We transplanted wild-type murine hearts into either wild-type or NOS2-null recipient mice; we then measured cardiac allograft survival and analyzed tissue sections by immunohistochemistry. We have confirmed that NOS2 increases cardiac allograft survival. We now show that there is less inflammation of cardiac allografts in wild-type hosts than in NOS2-null hosts. Furthermore, staining for von Willebrand factor reveals that the presence of NOS2 is correlated with the presence of Weibel-Palade bodies inside endothelial cells, whereas the absence of NOS2 is correlated with the release of Weibel-Palade bodies. CONCLUSIONS: Weibel-Palade bodies contain mediators that promote thrombosis and inflammation. Therefore, nitric oxide (NO) may stabilize the vessel wall and prevent endothelial activation in part by inhibiting the release of the contents of Weibel-Palade bodies. Prevention of Weibel-Palade body release might be a mechanism by which NO protects the vessel wall from inflammatory disorders such as atherosclerosis or graft arteriosclerosis.